Asymmetric bisaminoquinolines and bisaminoquinolines with varied linkers as autophagy inhibitors for cancer and other therapy

ABSTRACT

The invention provides novel asymmetric and symmetric bisaminoquinolmes and related compounds, methods of treatment and syntheses. The novel compounds exhibit effective anticancer activity and are useful in the treatment of a variety of autophagy-related disorders.

This application claims the benefit of priority of U.S. provisionalapplication Ser. No. 62/034,897, filed Aug. 8, 2014, of identical title,the entire contents of which are incorporated by reference herein.

RELATED APPLICATIONS AND GOVERNMENT INTEREST

The invention in this patent application was made with governmentsupport under Grant No. P01-CA114046 awarded by the National CancerInstitute. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention provides novel asymmetric and symmetric bisaminoquinolinesand related compounds, methods of treatment and syntheses. The novelcompounds exhibit unexpected anticancer activity and are useful in thetreatment of a variety of autophagy-related disorders.

BACKGROUND AND DESCRIPTION OF THE INVENTION

Autophagy consists of the sequestration of organelles and proteins inautophagic vesicles (AV) and degradation of this cargo through lysosomalfusion (1). Autophagy allows tumor cells to survive metabolic andtherapeutic stresses (2-5). Multiple publications indicatetherapy-induced autophagy is a key resistance mechanism to manyanti-cancer agents. Chloroquine (CQ) derivatives block autophagy byinhibiting the lysosome (3,6,7). Based on these findings, clinicaltrials combining cancer therapies with hydroxychloroquine (HCQ), (whichis safer than CQ to dose escalate) have been launched. Preliminaryresults indicate these combinations have activity (8-13), but it isstill unclear if this activity is consistently due to the addition ofHCQ. High micromolar concentrations of HCQ are required to inhibitautophagy. While there is some pharmacodynamic evidence of autophagyinhibition with HCQ in cancer patients, it is inconsistent becauseadequate concentrations are not achieved in all patients. There is anunmet need to develop more potent inhibitors of autophagy. The designand synthesis of dimeric analogs of CQ, that exploit the thermodynamicadvantages imparted by polyvalency (14, 15), has been a subject ofintensive study for over 10 years (16-18). An early report byVennerstrom (17) described the synthesis of heteroalkane-bridgedbisquinolines as potential antimalarials, but none of the compounds hadsufficient antimalarial activity to warrant further investigation.Subsequently, Sergheraert (16) reported that tetraquinolines, i.e.,dimers of bisquinolines, afforded potent antimalarials, confirming thepossibility that the application of the polyvalency strategy couldafford increased potency, at least with respect to antimalarialactivity.

More recently, Lee (19) has described the potentiation of AKT inhibitorsby fluorinated quinoline analogs. Solomon (20) has reported thepreparation of “repositioned” chloroquine dimers, based on the use of apiperazine connector. These results suggest that these chloroquineanalogs could serve as bases for the development of a new group ofeffective cancer chemotherapeutics. We have examined the application ofthe strategy of polyvalency (14, 15) to the synthesis of novel autophagyinhibitors by preparing a dimeric chloroquine (Lys01, FIG. 11 or 12),from commercially available materials. We have recently reported aseries of BAIs that potently inhibit autophagy and impair tumor growthin vivo (21). The structural motifs that are necessary for improvedautophagy inhibition compared to CQ include the presence of twoaminoquinoline rings and a triamine linker, as shown in the leadcompound, 1 (Lys 01) which is a 10-fold more potent autophagy inhibitorthan HCQ. Compared to HCQ, Lys 05, a water soluble salt of Lys01, morepotently accumulates within and deacidifies the lysosome, resulting inimpaired autophagy and tumor growth. At the highest dose administered,some mice developed Paneth cell dysfunction that resembles theintestinal phenotype of mice and humans with genetic defects in theautophagy gene ATG16L1(22), providing in vivo evidence that Lys05targets autophagy. Unlike HCQ, significant single agent antitumoractivity is observed without toxicity in mice bearing xenograft tumorstreated with lower doses of Lys05, establishing the therapeuticpotential of this compound in cancer. However, while Lys05 is 10-foldmore potent than HCQ in in vitro autophagy assays, it is cytotoxic onlyat micromolar concentrations in most cancer cells.

In the present invention, we demonstrate the preparation and theunexpected biological activity of asymmetric bisaminoquinolines andrelated compounds via changing the linker length and/or disrupting thesymmetry of the previously employed linkers. We describe unexpectedincrease in anti-cancer properties and capacity for autophagy inhibitionof bivalent aminoquinolines when linker length is changed substantiallyand/or asymmetrically from Lys01.

SUMMARY OF THE INVENTION

Herein we demonstrate the preparation and the unexpected biologicalactivity of asymmetric bis-4-aminoquinolines and related compounds bychanging the linker. We describe the unexpected increase in anti-cancerproperties of bivalent 4-aminoquinolines when linker length is changedsubstantially and asymmetrically from the parent Lys01 scaffold.

In one preferred embodiment, the invention provides a compound ofFormula IA:

wherein R¹ and R^(1′) are each independently H, F, Cl, Br, I, CN, NO₂,optionally substituted C₁-C₆ alkyl (when substituted, preferablysubstituted with 1 or 2 hydroxyl groups or 3-5 fluoro groups, preferablya CF₃ group), optionally substituted O—C₁-C₆ alkyl (preferably OCH₃),optionally substituted C₂-C₇ acyl (preferably acetyl), optionallysubstituted —(NH)-acyl, or optionally substituted C₂-C₇ ester(oxycarbonyl ester or carboxyester, preferably carboxyester) (each ofthe aforementioned groups, when substituted, is preferably substitutedwith one or two hydroxyl groups, one two or three halo groups or amethyl group)ss;R and R′ are each independently H, a C₁-C₆ optionally substituted alkylgroup, a C₁-C₇ (preferably C₂-C₇) optionally substituted acyl group, aC₂-C₇ optionally substituted carboxy ester group (which forms a urethanegroup with the nitrogen atom to which R or R′ is bonded);R″ is H, an amine protecting group (preferably benzoyl or benzyl), Cy¹or (C═O)_(z)-G, where Cy¹ is an optionally substituted cycloalkyl, arylor heteroaryl group, G is an optionally substituted C₀-C₁₂ (preferablyC₁-C₈, often C₁-C₃ alkyl) alkyl, alkene or alkynyl group (whereinoptional substituents include a C₁-C₁₂ (preferably C₁-C₈) alkyl, alkeneor alkynyl group substituted by (N—R^(J))—(C₁-C₈ alkyl, alkene oralkynyl group)_(z)-(Cy²)_(x), where R^(J) is H or a C₁-C₈ alkyl, alkeneor alkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is anoptionally substituted aryl or heteroaryl group (most preferably benzylor quinoline);R^(M) is independently at each occurrence H, F, Cl, Br, I, an optionallysubstituted C₁-C₁₂ (preferably C₁-C₈) alkyl, alkene, alkynyl or alkoxygroup (preferably including a CF₃ group);Each p in linker group L^(1A) is independently 1-10 (often 1, 2, 3, 4,5, 6, 7 or 8 or more often 1, 2 or 3) and optionally (preferably) each pin a molecule is different; and the pharmaceutically acceptable salts,enantiomers, diastereomers, solvates and polymorphs thereof, wherein thelength of designated linker group L^(1A) may vary, e.g. as illustratedin the compounds exemplified herein.

In another preferred embodiment, the invention provides a compound ofFormula IB:

or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvateor polymorph thereof, wherein the substituents are the same as definedfor Formula IA; andp and p′ in linker group L^(1B) are each independently 1, 2, 3, 4, 5, 6,7, 8, 9 or 10, often 1, 2, 3, 4, 5, or 6 and preferably, at least one ofp or p′ is different.

In another preferred embodiment, the invention provides a compound ofFormula IC:

or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvateor polymorph thereof, wherein the substituents are the same as definedfor Formula IA above, and each p in linker group L^(1C) is independently1-10 (often 1, 2, 3, 4, 5, 6, 7 or 8 or more often 1, 2 or 3) andoptionally (preferably) each p in a molecule is different

In another preferred embodiment, the invention provides a compound ofFormula ID:

or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvateor polymorph thereof, wherein the substituents are the same as definedfor Formula IA above and linker group L^(1D) is an optionallysubstituted alkylene group containing from 1-20, preferably 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 methylene groups, wherein one ormore methylene groups in linker group L^(1D) is preferably substitutedwith one or two hydroxyl groups, one or two halo groups or one or twoC₁-C₃ alkyl groups.

In still another embodiment, the present invention relates to compoundsaccording to the chemical structure IE:

Wherein R¹ and R^(1′) are each independently H, halo (F, Cl, Br or I),CN, NO₂, optionally substituted C₁-C₆ alkyl (when substituted,preferably substituted with 1 or 2 hydroxyl groups or 3-5 fluorogroups), optionally substituted O—C₁-C₆ alkyl (preferably, OCH₃),optionally substituted C₂-C₇ acyl (preferably acetyl), optionallysubstituted —(NH)-acyl, or optionally substituted C₂-C₇ ester(oxycarbonyl ester or carboxyester, preferably carboxyester);R_(M) is independently at each occurrence H, F, Cl, Br, I, an optionallysubstituted C₁-C₁₂ (preferably C₁-C₈) alkyl, alkene, alkynyl or alkoxygroup (preferably including a CF₃ group);R and R′ are each independently H, a C₁-C₆ optionally substituted alkylgroup, a C₁-C₇ (preferably C₂-C₇) optionally substituted acyl group, aC₂-C₇ optionally substituted carboxy ester group (which forms a urethanegroup with the nitrogen atom to which R or R′ is bonded);Linker group L^(1E) is a

group or a

group (either A or A′ may be bonded to either of the two amine groups incompound I) wherein at least one of the CH₂ groups in L is optionallysubstituted with an optionally substituted C₁-C₆ alkyl group includingan optionally substituted cyclic alkyl group which, when substituted,are preferably substituted with one or two hydroxyl groups, one two orthree halo groups (preferably Cl or F) or one or two C₁-C₃ alkyl groups(preferably methyl), or at least two methylene groups in linker group L(preferably two adjacent methylene groups) are substituted with alkylenegroups to form an optionally substituted C₃-C₈ (preferably a C₅-C₆)cycloalkyl group (ring), which itself, when substituted, is preferablysubstituted with one or two hydroxyl groups, one two or three halogroups (preferably F or Cl) or one or two C₁-C₃ alkyl groups (preferablymethyl), or linker group L is a (poly)ethylene glycol group having from1 to 20 (preferably 2 to 15, 3 to 10 or 4, 5, 6, 7, 8, 9, or 10)ethylene glycol units, each (poly)ethylene glycol group being optionallysubstituted at one or both of its distal ends with an A group, or linkergroup L is a linear group containing from 1 to 5 non-contiguous aminegroups N—R″, each amine group being separated from an adjacent aminegroup by an alkylene group containing from 1, 2, 3, 4, 5, 6, 7, or 8(preferably 2, 3, 4, 5 or 6) methylene groups or an ethylene glycolcontaining group having from 1 to 6 (preferably 2 to 6, 3 to 6 or 4, 5or 6) ethylene glycol units;X is absent, (CH₂)_(j), O, S or N—R″;Y is absent, CH₂, O, CH₂O or N—R″ and Y′ is absent CH₂, O, OCH₂ or N—R″,with the proviso that when one or more of X, Y and Y′ is present, eachof X and Y, X and Y′ or Y and Y′, when present, forms a bond;R″ is H or an optionally substituted C₁-C₆ (preferably C₁-C₃) alkylgroup or an amine protecting group (preferably H, Me, benzoyl orbenzyl);each j is independently 1, 2, 3, 4, 5 or 6 (preferably 1 or 2);each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 with theproviso that when n is 0, X is (CH₂) where j is at least 1 and at leastone CH₂ group is optionally substituted, preferably with a C₁-C₃ alkylgroup which itself is optionally substituted with one or two hydroxylgroups or one two or three halo groups;A is absent, C═O or (CH₂) and A′ is C═O or (CH₂)_(j) wherein at leastone CH₂ group in A or A′ is optionally substituted, preferably with aC₁-C₃ alkyl group which is itself optionally substituted, preferablywith one or two hydroxyl groups or one, two or three halo groups;

Z is O or N—R^(Z);

R^(Z) is H or an optionally substituted C₁-C₃ alkyl group,or a pharmaceutically acceptable salt, enantiomer, diastereomer, solventor polymorph thereof.

In certain preferred embodiments of the invention, at least one(preferably two) R^(M) is other than H, especially when R¹ and R^(1′)are H. In other preferred aspects of the invention, R¹ and R^(1′) areeach independently H, a halo group (most often Cl or F), a nitro groupor a trifluoromethyl group, most often a chloro or fluoro group. R andR′ are preferably each independently H or methyl, preferably H and L ispreferably an alkylene group containing from 6 to 20 methylene groups(6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19 or 20), a(poly)ethylene glycol group having from 1 to 20 (1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 2 to 15, 3to 10 or 4, 5, 6, 7, 8, 9, or 10) ethylene glycol units, each(poly)ethylene glycol group being optionally substituted at one or bothof its distal ends with an A group, or L is a linear group containingfrom 1 to 5 non-contiguous amine groups N—R″, each amine group beingseparated from an adjacent amine group by an alkylene group containingfrom 1, 2, 3, 4, 5, 6, 7, or 8 (preferably 2, 3 or 4) methylene groupsor an ethylene glycol containing group having from 1 to 6 (preferably 2to 6, 3 to 6 or 4, 5 or 6) ethylene glycol units. R″ is preferably H,methyl or an amine protecting group (preferably a benzyl or benzoylgroup which can be removed to introduce other groups on the amine groupafter deprotection).

In certain embodiments, one or more amine group of L is optionallysubstituted with one or two 7-substituted-4-quinolinyl group(s) whereinthe amine binds to the 4-position of the quinolinyl group through analkylene group and the 7-position of each quinolinyl group is optionallysubstituted, preferably with a R¹ and/or R^(1′) group as broadlydescribed for generic structure I above, or one or more amines of L issubstituted with an alkyl group which itself is further optionallysubstituted with at least one hydroxyl group, an alkoxy group, an amine,a monoalkyl (C₁-C₆, preferably C₁-C₃) amine or a dialkyl (C₁-C₆,preferably C₁-C₃) amine wherein the amine or monoalkyl amine isoptionally substituted on the amine position with one or two7-substituted-quinolinyl group(s) wherein the amine binds to the4-position of the quinolinyl group and the 7-position of each quinolinylgroup is optionally substituted, preferably with R¹ and/or R^(1′) asbroadly described for generic structure I above, and each of said alkoxygroups (e.g. methoxy or ethoxy) is optionally further substituted with aC₁-C₃ alkosssxy group, preferably a methoxy group, thus forming adiether substituent.

In certain embodiments of the invention, R¹ and R^(1′) are eachindependently H, a halo group, a nitro group or a trifluoromethyl group,preferably a chloro or fluoro group. R and R′ are preferably eachindependently H or a C₁-C₃ optionally substituted alkyl group itselfpreferably substituted with at least one hydroxyl group, an amine,monoalkyl amine or dialkyl amine (where one or both alkyl groups isitself further optionally substituted with a dialkyl amine or an aminesubstituted with one or two (preferably one) 7-substituted-4-quinolinylgroup(s) where the amine group is bonded to the 4-position of thequinolinyl group), an alkoxy group (e.g. methoxy or ethoxy) which may befurther substituted with an alkoxy group, preferably a methoxy group(thus forming a diether substituent).

In a particularly preferred embodiment, the invention provides compoundsof the formulae IIA, JIB, IIC and IID, and the pharmaceuticallyacceptable salts, enantiomers, diastereomers, solvates or polymorphsthereof:

where X is a halogen (preferably F or Cl), ora pharmaceutically acceptable salt thereof;

where X is a halogen (preferably F or Cl), ora pharmaceutically acceptable salt thereof;

where X is a halogen (preferably F or Cl), ora pharmaceutically acceptable salt thereof; and

where X is a halogen (preferably F or Cl), ora pharmaceutically acceptable salt thereof.

Further preferred compounds according to the present invention includethose which are presented or identified in any of FIGS. 10-13 or thevarious schemes as presented herein.

In other embodiments, the invention provides methods of treatment andpharmaceutical compositions which use therapeutically-effective amountsof compounds of Formulae IA, IB, IC, ID, IE, IIA, IIB and IIC, the othercompounds described herein (e.g. the compounds of Tables 1-2 and FIGS.10-13) and the pharmaceutically acceptable salts, enantiomers,diastereomers, solvents or polymorphs thereof, to treat cancer and otherautophagy-related disorders.

As described further hereinafter, compounds of the invention exhibit anunexpectedly high level of cytotoxicity in a variety of cancer cells andevidence effective autophagy inhibition at surprising low doses,indicating their effectiveness in the treatment of broad spectrum ofautophagy-related disorders.

These and other aspects of the invention are illustrated further in theDetailed Description of the Invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the cytotoxicity of dimeric CQs with longer linkers. (A)IC50 of selected Lys01 derivatives (complete dataset in table1) withlonger linkers in a 72 hour alamar blue viability assay. A375 P cellsseeded in a 384 well format were treated with a 10 point dilution ofconcentrations (0.01 nM-10 uM) (B) Colony formation assay in soft agar.Cells from the indicated melanoma (A375, WM983B, SKMEL5) and pancreaticcancer (PANC1, CAPAN1) cell lines were plated in soft agar. Medium withcompound was changed every 48 hours. Crystal violet staining was used tovisualize formed colonies after 2 weeks.

FIG. 2 shows the effects of asymmetry and longer linker length on thecytotoxicity of dimeric chloroquines. A375P melanoma cells were platedin a 384 well format and treated with increasing concentrations of Lys01derivatives. After 72 hours the cells were analyzed by alamar blue.Complete data is in Table1, FIG. 10. (A) IC50 according to symmetry oflinker (B) IC50 by linker length.

FIG. 3 shows subcellular localization of dimeric chloroquines withvaried linkers. (A) Lys21 was tagged with a fluorescent dye Cy3 (B)A375P melanoma cells stained with lysotracker (light grey), andco-treated with Lys21-Cy3. Lighter color in third frame indicatescolocalization.

FIG. 4 shows autophagy modulation of dimeric chloroquines with longerlinkers in melanoma cell lines. LC3B and actin immunoblotting in 3different melanoma cell lines. Each compound was administered at 1 uMconcentrations for 24 hours. Quantification of LC3II/actin indicates alldimeric CQs with longer linker produced higher levels of LC3II/actincompared with Lys05 (dotted line).

FIG. 5 shows high throughput phenotypic screen of autophagy inhibitionfor Lys01 derivatives. A375PmCherry-eGFP-LC3 cells were seeded in 384well plates. Doxorubicin, HCQ, CQ and Lys01-Lys27 (in 0.1% DMSO) wereadministered through robotic assisted pin tool transfer atconcentrations of 0-10 μM. Images were obtained by the Operettahigh-content fluorescent microscopy. Image analysis using Harmonysoftware reports fraction of nucleated cells with ratio of >3.5 in cellstreated with 10 μM compound.

FIG. 6 shows high throughput fluorescence microscopy images of A375PmCherry-eGFP-LC3 melanoma cells treated with indicated chloroquinederivatives.

FIG. 7 shows histograms of green/red ratio in Lys01-derivative treatedA375PmCherry-GFP-LC3 cells.

FIG. 8 shows flow cytometry quantification of reactive oxygen species(ROS), autophagy inhibition and apoptosis associated with dimeric CQswith longer linkers. (A) ROS reflected by DHR_123 fluorescence in A375cells after 24 hours of treatment with luM compound (B) Autophagyinhibition reflected by green/red ratio of A375 mCherry-egfpLC3 cellstreated with luM indicated compound for 24 hours. (C) Apoptosisreflected by annexin V detection in A375 cells treated with luM compoundat the 24 hours. (D) Apoptosis reflected by annexin V detection in A375cells treated with indicated compounds at 3 uM for 24 or 48 hours. PLX:PLX4720; TRA: trametinib.

FIG. 9 shows the effects of dimeric CQ's with longer linker on growthand invasion in a 3D tissue like culture model. A375P mCherry eGFP-LC3cells grown into 3 dimensional spheroids and implanted in collagen.After 72 hours of treatment with 3 uM of indicated compound, cells werecounterstained with DAPI. (A) fluorescence microscopy was used tovisualize autophagy inhibition (bright areas), inhibition of invasioninto the collagen and (B) quantification of the DAPI fluorescenceindicative of cell death. * p<0.05

FIG. 10, Table 1 shows the results of cytotoxicity experiments usingcompounds according to the present invention. LN229 glioma cells, andA375P melanoma cells were plated in a 384 well format and compounds CQ,HCQ, and Lys01-Lys41 and 72-75 (IUPAC names and chemical structuresprovided in FIGS. 11 and 12) were delivered in concentrations between0.01-10 micromolar using robotic assisted dispenser. After 72 hoursincubation at 37 degrees, Alamar blue was applied and viability wasdetermined using absorbance. Absorbance was normalized to DMSO control,and a log IC50 was estimated using Graphpad Prism software.

FIG. 11, Table 2 provides the IUPAC names for the compounds which areset forth in attached FIG. 12.

FIG. 12 shows representative chemical structures of compounds accordingto the present invention.

FIG. 13 shows the chemical structures of a number of novelbisaminoquinolines according to the present invention with varioushalo-substituents (F, Cl, Br) on the 7-position of the quinoline moiety.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used throughout the specification todescribe the present invention. Where a term is not specifically definedherein, that term shall be understood to be used in a manner consistentwith its use by those of ordinary skill in the art.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges that may independently be included in thesmaller ranges are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention. In instanceswhere a substituent is a possibility in one or more Markush groups, itis understood that only those substituents which form stable bonds areto be used.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, generally a mammal, especiallyincluding a domesticated animal and preferably a human, to whomtreatment, including prophylactic treatment (prophylaxis), with thecompounds or compositions according to the present invention isprovided. For treatment of those infections, conditions or diseasestates which are specific for a specific animal such as a human patient,the term patient refers to that specific animal. In most instances, thepatient or subject of the present invention is a human patient of eitheror both genders.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount of a compound or component which, when used withinthe context of its use, produces or effects an intended result, whetherthat result relates to the prophylaxis and/or therapy of an infectionand/or disease state or as otherwise described herein. The termeffective subsumes all other effective amount or effective concentrationterms (including the term “therapeutically effective”) which areotherwise described or used in the present application.

The term “compound” is used herein to describe any specific compound orbioactive agent disclosed herein, including any and all stereoisomers(including diasteromers), individual optical isomers (enantiomers) orracemic mixtures, pharmaceutically acceptable salts and prodrug forms.The term compound herein refers to stable compounds. Within its use incontext, the term compound may refer to a single compound or a mixtureof compounds as otherwise described herein. It is understood that thechoice of substituents or bonds within a Markush or other group ofsubstituents or bonds is provided to form a stable compound from thosechoices within that Markush or other group. In compounds according tothe present invention, R¹ and R^(M) or R^(1′) and R^(M) do not togetherform a cyclic ring.

The term “bioactive agent” refers to any biologically active compound ordrug which may be formulated for use in the present invention. Exemplarybioactive agents include the compounds according to the presentinvention which are used to inhibit autophagy and to treat cancer aswell as other compounds or agents which are otherwise described herein.

The terms “treat”, “treating”, and “treatment”, are used synonymously torefer to any action providing a benefit to a patient at risk for orafflicted with a disease, including improvement in the condition throughlessening or suppression of at least one symptom, delay in progressionof the disease, prevention or delay in the onset of the disease, etc.

Treatment, as used herein, encompasses both prophylactic and therapeutictreatment, principally of cancer. Compounds according to the presentinvention can, for example, be administered prophylactically to a mammalin advance of the occurrence of disease to reduce the likelihood of thatdisease. Prophylactic administration is effective to reduce or decreasethe likelihood of the subsequent occurrence of disease in the mammal, ordecrease the severity of disease that subsequently occurs, especiallyincluding metastasis of cancer. Alternatively, compounds according tothe present invention can, for example, be administered therapeuticallyto a mammal that is already afflicted by disease. In one embodiment oftherapeutic administration, administration of the present compounds iseffective to eliminate the disease and produce a remission orsubstantially eliminate the likelihood of metastasis of a cancer.Administration of the compounds according to the present invention iseffective to decrease the severity of the disease or lengthen thelifespan of the mammal so afflicted, in the case of cancer.

The term “pharmaceutically acceptable” as used herein means that thecompound or composition is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the severity of the disease and necessity of thetreatment.

The term “inhibit” as used herein refers to the partial or completeelimination of a potential effect, while inhibitors are compounds thathave the ability to inhibit.

The term “prevention” when used in context shall mean “reducing thelikelihood” or preventing a disease, condition or disease state fromoccurring as a consequence of administration or concurrentadministration of one or more compounds or compositions according to thepresent invention, alone or in combination with another agent. It isnoted that prophylaxis will rarely be 100% effective; consequently theterms prevention and reducing the likelihood are used to denote the factthat within a given population of patients or subjects, administrationwith compounds according to the present invention will reduce thelikelihood or inhibit a particular condition or disease state (inparticular, the worsening of a disease state such as the growth ormetastasis of cancer) or other accepted indicators of diseaseprogression from occurring.

The term “autophagy” or “autophagocytosis” is used to describe acatabolic process in cells which involves the degradation of a cell'sown components through lysosomes. Autophagy is a highly regulatedprocess of biological systems that plays a normal part in cell growthdevelopment and homeostasis. helping to maintain a balance between thesynthesis, degradation, and subsequent recycling of cellular products.It is a major mechanism by which a cell allocates nutrients fromunnecessary processes to more-essential processes.

A number of autophagic processes occur in nature, all of which have thedegradation of intracellular components via the lysosome as a commonfeature. A well-known mechanism of autophagy involves the formation of amembrane around a targeted region of a cell, separating the contentsfrom the rest of the cytoplasm. The resultant vesicle then fuses with alysosome which subsequently degrades the contents.

Autophagy consists of the sequestration of organelles and proteins inautophagic vesicles (AV) and degradation of this cargo through lysosomalfusion (1). Autophagy allows tumor cells to survive metabolic andtherapeutic stresses (2-5). Multiple publications indicatetherapy-induced autophagy is a key resistance mechanism to manyanti-cancer agents.

An “autophagy-related disorder” includes diseases, disease states and/orconditions which benefit from the inhibition of autophagy, including,but not limited to, cancer (including the metastasis of cancer),rheumatoid arthritis, malaria, antiphospholipid antibody syndrome,lupus, chronic urticaria and Sjogren's disease.

The term “cancer” shall refer to a proliferation of tumor cells havingthe unique trait of loss of normal controls, resulting in unregulatedgrowth, lack of differentiation, local tissue invasion, and/ormetastasis. As used herein, neoplasms include, without limitation,morphological irregularities in cells in tissue of a subject or host, aswell as pathologic proliferation of cells in tissue of a subject, ascompared with normal proliferation in the same type of tissue.Additionally, neoplasms include benign tumors and malignant tumors(e.g., colon tumors) that are either invasive or noninvasive. Malignantneoplasms are distinguished from benign neoplasms in that the formershow a greater degree of dysplasia, or loss of differentiation andorientation of cells, and have the properties of invasion andmetastasis. The term cancer also within context, includes drug resistantcancers, including multiple drug resistant cancers, metastatic cancersand/or recurrent cancers. Examples of neoplasms or neoplasias from whichthe target cell of the present invention may be derived include, withoutlimitation, carcinomas (e.g., squamous-cell carcinomas, adenocarcinomas,hepatocellular carcinomas, and renal cell carcinomas), particularlythose of the bladder, bone, bowel, breast, cervix, colon (colorectal),esophagus, head, kidney, liver, lung, nasopharyngeal, neck, thyroid,ovary, pancreas, prostate, and stomach; leukemias, such as acutemyelogenous leukemia, acute lymphocytic leukemia, acute promyelocyticleukemia (APL), acute T-cell lymphoblastic leukemia, adult T-cellleukemia, basophilic leukemia, eosinophilic leukemia, granulocyticleukemia, hairy cell leukemia, leukopenic leukemia, lymphatic leukemia,lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic leukemia,micromyeloblastic leukemia, monocytic leukemia, neutrophilic leukemiaand stem cell leukemia; benign and malignant lymphomas, particularlyBurkitt's lymphoma, Non-Hodgkin's lymphoma and B-cell lymphoma; benignand malignant melanomas; myeloproliferative diseases; sarcomas,particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma,liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovialsarcoma; tumors of the central nervous system (e.g., gliomas,astrocytomas, oligodendrogliomas, ependymomas, gliobastomas,neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas,pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, andSchwannomas); germ-line tumors (e.g., bowel cancer, breast cancer,prostate cancer, cervical cancer, uterine cancer, lung cancer (e.g.,small cell lung cancer, mixed small cell and non-small cell cancer,pleural mesothelioma, including metastatic pleural mesothelioma smallcell lung cancer and non-small cell lung cancer), ovarian cancer,testicular cancer, thyroid cancer, astrocytoma, esophageal cancer,pancreatic cancer, stomach cancer, liver cancer, colon cancer, andmelanoma; mixed types of neoplasias, particularly carcinosarcoma andHodgkin's disease; and tumors of mixed origin, such as Wilms' tumor andteratocarcinomas, among others. It is noted that certain epithelialtumors including ovarian, breast, colon, head and neck, medulloblastomaand B-cell lymphoma, among others are shown to exhibit increasedautophagy and are principal target cancers for compounds and therapiesaccording to the present invention.

The term “additional anti-cancer agent” is used to describe anadditional compound which may be coadministered with one or morecompounds of the present invention in the treatment of cancer. Suchagents include, for example, everolimus, trabectedin, abraxane, TLK 286,AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244(ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, aFLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurorakinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDACinhbitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFRTK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinaseinhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek)inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib,nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu,nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin,tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab,ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490,cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR₁ KRX-0402,lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102,talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib,5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin,irinotecan, liposomal doxorubicin, 5′-deoxy-5-fluorouridine,vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244,capecitabine, L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258);3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6, Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂)_(X) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662,tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid,valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951,aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BacillusCalmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevac,hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole,lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna,methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide,oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, teniposide,testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine,13-cis-retinoic acid, phenylalanine mustard, uracil mustard,estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosinearabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin,mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat,COL-3, neovastat, BMS-275291, squalamine, endostatin, SU541.6, SU6668,EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene,idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,denileukin diftitox, gefitinib, bortezimib, paclitaxel, irinotecan,topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonalantibody) and erbitux, cremophor-free paclitaxel, epithilone B,BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene,ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene,TSE-424, HMR-3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352,rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573,RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684,LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating f_(actor), zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, sspegfilgrastim, erythropoietin,epoetin alfa and darbepoetin alfa, ipilumumab, vemurafenib among others.

The term “alkyl” is used herein to refer to a fully saturated monovalentradical containing carbon and hydrogen (up to 10 carbon atoms or asotherwise indicated), and which may be a straight chain, branched orcyclic. Examples of alkyl groups are methyl, ethyl, n-butyl, n-heptyl,isopropyl, 2-methyl propyl, tert-butyl, neopentyl, hexyl, heptyl, octyl,nonyl, decyl, etc.

The term “substituted” as that term relates to alkyl groups which aredescribed above include one or more functional groups such as loweralkyl groups containing 1-6 carbon atoms which are optionallysubstituted with 1 or 2 hydroxyl groups or between 1 and 5 (preferably3-5) fluoro groups, acyl (C₁-C₆), halogen (F, Cl, Br, I, e.g., alkylhalos, e.g., CF₃), amido, hydroxyl, carboxy/carboxylic acid, thioamido,cyano, nitro, alkenyl (C₂-C₆) alkynyl (C₂-C₆), azido, alkoxy (C₁-C₆),(including alkoxy groups which are further substituted with a C₁-C₆alkoxy group thus producing a diether group), amino, C₁-C₆ alkylaminoand dialkyl-amino, where the alkyl groups may be optionally substitutedwith 1 or 2 hydroxyl groups or an amine, aminoalkyl or dialkyl groupwhich itself is substituted one or two alkyl groups or a7-substituted-4-quinolinyl group, C₂-C₆ acylamino, C₂-C₆ oxyacylester orcarboxyester, aryloxy, aryloxy(C₁-C₆)alkyl, carboxamido, thio, C₂-C₆ether or thioether, a 7-substituted-4-aminoquinolinyl group (or asubstitution on an amine group which forms a7-substituted-4-aminoqunolinyl group) and the like. Preferredsubstituents on alkyl groups (within context, especially on the aminogroup of the 7-substituted-4-aminoquinoline) or a linker which containsat least one amine group, include, for example, at least one hydroxylgroup, an amine, monoalkyl amine or dialkyl amine (where one or bothalkyl groups is itself further optionally substituted with a dialkylamine or an amine substituted with one or two (preferably one)7-substituted-4-quinolinyl group(s) where the amine group is bonded tothe 4-position of the quinolinyl group) or an alkoxy group (e.g. methoxyor ethoxy) which may be further substituted with an alkoxy group,preferably a methoxy group, thus forming a diether substituent.

The term “aryl” refers to a substituted or unsubstituted monovalentaromatic radical having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl). Other examples include heterocyclicaromatic (heteroaromatic or heteroaryl) ring groups having one or morenitrogen, oxygen, or sulfur atoms in the ring, in particular, quinolinegroups, in particular, 7-substituted-amino quinoline groups, as well asother groups.

The term “substituted” as used in the term “substituted aryl,substituted aromatic, substituted heteroaryl, or substitutedheteroaromatic” herein signifies that a substitution on the 7-positionof 4-aminoquinoline may be present, said substituents being selectedfrom atoms and groups, which when present enhance the activity of thecompound as an inhibitor of autophagy. Examples of substituents that maybe present in a substituted aromatic or heteroaromatic group include,but are not limited to, groups such as H, halo (F, Cl, Br or I), CN,NO₂, optionally substituted C₁-C₆ alkyl (when substituted, preferablysubstituted with 1 or 2 hydroxyl groups or 3-5 fluoro groups),optionally substituted O—C₁-C₆ alkyl (preferably, OCH₃), optionallysubstituted C₂-C₇ acyl (preferably acetyl) or optionally substitutedC₂-C₇ ester (oxycarbonyl ester or carboxyester, preferablycarboxyester). It is noted that each of the substituents disclosedherein may themselves be substituted.

The term “co-administration” or “adjunct therapy” shall mean that atleast two compounds or compositions are administered to the patient atthe same time, such that effective amounts or concentrations of each ofthe two or more compounds may be found in the patient at a given pointin time. Although compounds according to the present invention may beco-administered to a patient at the same time, the term embraces bothadministration of two or more agents at the same time or at differenttimes, including sequential administration. Preferably, effectiveconcentrations of all co-administered compounds or compositions arefound in the subject at a given time. The term co-administration oradjunct therapy also contemplates other bioactive agents beingcoadministered with pharmaceutical compositions according to the presentinvention, especially where a cancer has metastasized or is at risk formetastasis.

The term “amine protecting group” refers to a moiety or group which canbe readily removed from a functional group to which the protecting groupis attached to allow further reaction. Exemplary amine-protecting groupsinclude carbobenzyloxy (Cbz group, removed by hydrogenolysis),p-Methoxylbenzyl carbon (Moz or MeOZ group, removed by hydrogenolysis),tert-butyloxycarbonyl (BOC group, removed by concentrated strong acid orby heating at elevated temperatures), 9-Fluorenylmethyloxycarbonyl (FMOCgroup, removed by weak base, such as piperidine or pyridine), acyl group(acetyl, benzoyl, pivaloyl, by treatment with base), benzyl (Bn groups,removed by hydrogenolysis), carbamate, removed by acid and mild heating,p-methoxybenzyl (PMB, removed by hydrogenolysis), 3,4-dimethoxybenzyl(DMPM, removed by hydrogenolysis), p-methoxyphenyl (PMP group, removedby ammonium cerium IV nitrate or CAN); tosyl (Ts group removed byconcentrated acid and reducing agents, other sulfonamides, Mesyl, Nosyl& Nps groups, removed by samarium iodide, tributyl tin hydride.

The term “radiotherapy” or “radiation therapy” is used to describetherapy for cancer which may be used in conjunction with the presentcompounds. Radiation therapy uses high doses of radiation, such asX-rays, or other energy sources such as radioisotopes (gamma, beta oralpha emitters), to destroy cancer cells. The radiation damages thegenetic material of the cells so that they can't grow. Althoughradiation damages normal cells as well as cancer cells, the normal cellscan repair themselves and function, while the cancer cells cannot.

Radiation therapy may be used in combination with the presently claimedcompounds, alone or in combination with additional anticancer compoundsas otherwise disclosed herein, depending on the cancer to be treated.Radiotherapy therapy is most effective in treating cancers that have notspread outside the area of the original cancer, but it also may be usedif the cancer has spread to nearby tissue. Radiotherapy is sometimesused after surgery to destroy any remaining cancer cells and to relievepain from metastatic cancer.

Pharmaceutical Compositions

Compounds according to the present invention may be readily formulatedinto pharmaceutical compositions, useful in the inhibition of autophagyin a biological system and/or the inhibition, treatment or prevention ofdiseases states and/or conditions which benefit from the inhibition ofautophagy including cancer (and its metastasis), rheumatoid arthritis,malaria, antiphospholipid antibody syndrome, lupus (systemic lupuserythematosus), chronic urticaria and Sjogren's disease. Pharmaceuticalcompositions comprise an effective amount of one or more compoundsaccording to the present invention in combination with apharmaceutically acceptable carrier, additive or excipient, optionallyin combination with at least one additional agent, in the case ofcancer, preferably an anticancer agent as otherwise described herein.

As noted above, the compounds and method of the invention may be used toinhibit autophagy as otherwise described herein, and are useful for theinhibition (including prophylaxis) and/or treatment of cancer and itsmetastasis, rheumatoid arthritis, malaria, antiphospholipid antibodysyndrome, lupus (systemic lupus erythematosus), chronic urticaria andSjogren's disease. The treatment of cancer or malaria are importantaspects of the present invention.

In methods according to the present invention, subjects or patients inneed are treated with the present compounds, pharmaceutical compositionsin order to inhibit, reduce the likelihood or treat a disease state,condition and/or infection as otherwise described herein. The diseasestates, conditions and infections treated by the present compounds andcompositions are readily recognized and diagnosed by those of ordinaryskill in the art and treated by administering to the patient aneffective amount of one or more compounds according to the presentinvention.

Generally, dosages and routes of administration of the compound aredetermined according to the size and condition of the subject, accordingto standard pharmaceutical practices. Dose levels employed can varywidely, and can readily be determined by those of skill in the art.Typically, amounts in the milligram up to gram quantities are employed.The composition may be administered to a subject by various routes, e.g.orally, transdermally, perineurally or parenterally, that is, byintravenous, subcutaneous, intraperitoneal, itrathecally orintramuscular injection, among others, including buccal, rectal andtransdermal administration. Compositions may also be administered byinhalation to the lungs. Subjects contemplated for treatment accordingto the method of the invention include humans, companion animals,laboratory animals, and the like.

Formulations containing the compounds according to the present inventionmay take the form of solid, semi-solid, lyophilized powder, or liquiddosage forms, such as, for example, tablets, capsules, powders,sustained-release formulations, solutions, suspensions, emulsions,suppositories, creams, ointments, lotions, aerosols, patches or thelike, preferably in unit dosage forms suitable for simple administrationof precise dosages.

Pharmaceutical compositions according to the present invention typicallyinclude a conventional pharmaceutical carrier or excipient and mayadditionally include other medicinal agents, carriers, adjuvants,additives and the like. Preferably, the composition is about 0.1% toabout 85%, about 0.5% to about 75% by weight of a compound or compoundsof the invention, with the remainder consisting essentially of suitablepharmaceutical excipients. For oral administration, such excipientsinclude pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, glucose, gelatin,sucrose, magnesium carbonate, and the like. If desired, the compositionmay also contain minor amounts of non-toxic auxiliary substances such aswetting agents, emulsifying agents, or buffers.

Liquid compositions can be prepared by dissolving or dispersing thecompounds (about 0.5% to about 20% by weight or more), and optionalpharmaceutical adjuvants, in a carrier, such as, for example, aqueoussaline, aqueous dextrose, glycerol, or ethanol, to form a solution orsuspension. For use in oral liquid preparation, the composition may beprepared as a solution, suspension, emulsion, or syrup, being suppliedeither in liquid form or a dried form suitable for hydration in water ornormal saline.

When the composition is employed in the form of solid preparations fororal administration, the preparations may be tablets, granules, powders,capsules or the like. In a tablet formulation, the composition istypically formulated with additives, e.g. an excipient such as asaccharide or cellulose preparation, a binder such as starch paste ormethyl cellulose, a filler, a disintegrator, and other additivestypically used in the manufacture of medical preparations.

An injectable composition for parenteral administration will typicallycontain the compound in a suitable i.v. solution, such as sterilephysiological salt solution. The composition may also be formulated as asuspension in a lipid or phospholipid, in a liposomal suspension, or inan aqueous emulsion.

Methods for preparing such dosage forms are known or is apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences (17th Ed., Mack Pub. Co., 1985). The composition to beadministered will contain a quantity of the selected compound in apharmaceutically effective amount for inhibiting autophagy in abiological system, including a patient or subject according to thepresent invention.

Method of Treatment

According to one aspect of the invention, a method is provided fortreating a mammalian patient or subject to inhibit autophagy in thatpatient or subject. Compounds according to the present inventiondescribed herein may be used to inhibit autophagy in a manner consistentwith inhibiting, treating and/or preventing disease states and/orconditions including cancer (including metastasis of cancer), rheumatoidarthritis, malaria, antiphospholipid antibody syndrome, lupus, chronicurticaria and Sjogren's disease.

According to the present invention, in patients or subjects in needthereof, are treated by administering to the patient or subject aneffective amount of one or more compounds according to the presentinvention, optionally in combination with at least one additionalbioactive agent useful for treating the same disease state or condition.Compounds according to the present invention may be used to inhibit,reduce the likelihood or treat cancer, including the metastasis ofcancer in a patient or subject in need of such treatment. The treatmentis useful for any cancer for which inhibition of autophagy represents afavorable result or for which metastasis is a risk element. Therapy withat least one additional anticancer agent as otherwise described hereinis also contemplated in the present methods. The numerous cancers whichmay be treated pursuant to the present method are described hereinabove.

In another aspect the present invention is directed to a method fortreating a disease state and/or condition which benefits from theinhibition of autophagy, including rheumatoid arthritis, malaria,antiphospholipid antibody syndrome, lupus, chronic urticaria andSjorgen's disease. In this method, a patient or subject in need oftreatment is administered an effective amount of a compound as otherwisedescribed herein optionally in combination with a pharmaceuticallyacceptable carrier, additive or excipient in order to inhibit, treatand/or prevent the above disease states of conditions. In alternativeembodiments, at least one additional bioactive agent is coadministeredwith a compound according to the present invention.

In certain preferred embodiments of the invention, at least one(preferably two) R^(M) is other than H, especially when R¹ and R^(1′)are H. In other preferred aspects of the invention, R¹ and R^(1′) areeach independently H, a halo group (most often Cl or F), a nitro groupor a trifluoromethyl group, most often a chloro or fluoro group. R andR′ are preferably each independently H or methyl, preferably H and L ispreferably an alkylene group containing from 6 to 20 methylene groups(6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 18, 19 or 20), a(poly)ethylene glycol group having from 1 to 20 (1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 2 to 15, 3to 10 or 4, 5, 6, 7, 8, 9, or 10) ethylene glycol units, each(poly)ethylene glycol group being optionally substituted at one or bothof its distal ends with an A group, or L is a linear group containingfrom 1 to 5 non-contiguous amine groups N—R″, each amine group beingseparated from an adjacent amine group by an alkylene group containingfrom 1, 2, 3, 4, 5, 6, 7, or 8 (preferably 2, 3 or 4) methylene groupsor an ethylene glycol containing group having from 1 to 6 (preferably 2to 6, 3 to 6 or 4, 5 or 6) ethylene glycol units. R″ is preferably H,methyl or an amine protecting group (preferably a benzyl or benzoylgroup which can be removed to introduce other groups on the amine groupafter deprotection).

In certain embodiments, one or more amine group of L is optionallysubstituted with one or two 7-substituted-4-quinolinyl group(s) whereinthe amine binds to the 4-position of the quinolinyl group through analkylene group and the 7-position of each quinolinyl group is optionallysubstituted, preferably with a R¹ and/or R^(1′) group as broadlydescribed for generic structures IA, IB, IC, ID and IE above, or one ormore amines of the linker group is substituted with an alkyl group whichitself is further optionally substituted with at least one hydroxylgroup, an alkoxy group, an amine, a monoalkyl (C₁-C₆, preferably C₁-C₃)amine or a dialkyl (C₁-C₆, preferably C₁-C₃) amine wherein the amine ormonoalkyl amine is optionally substituted on the amine position with oneor two 7-substituted-quinolinyl group(s) wherein the amine binds to the4-position of the quinolinyl group and the 7-position of each quinolinylgroup is optionally substituted, preferably with R¹ and/or R^(1′) asbroadly described for generic structures IA, IB, IC, ID and IE above,and each of said alkoxy groups (e.g. methoxy or ethoxy) is optionallyfurther substituted with a C₁-C₃ alkoxy group, preferably a methoxygroup, thus forming a diether substituent.

Further preferred methods relate to the use/administration of thecompounds according to the present invention which are presented in thevarious schemes which are presented in the tables and figures presentedherein.

In the methods treating or inhibiting cancer or the metastasis ofcancer, the compounds described above may be coadministered with atleast one additional anticancer agent including, for example,everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib,GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107,TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457,MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFRinhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1modulator, a Bcl-2 inhibitor, an HDAC inhbitor, a c-MET inhibitor, aPARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TKinhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKTinhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focaladhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGFtrap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib,panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171,batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan,tesmilifene, oblimersen, ipilimumab, gossypol, Bio 111, 131-I-TM-601,ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO1001, IPdR₁ KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta744, Sdx 102, talampanel, atrasentan, Xr 311 romidepsin, ADS-100380,sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine,doxorubicin, irinotecan, liposomal doxorubicin,5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]jbenzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258);3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6, Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂)_(X) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PM-166, GW-572016, Ionafarnib, BMS-214662, tipifarnib;amifostine, NVP-LAQ824, suberoyl anilide hydroxamic acid, valproic acid,trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide,arnsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG)vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, cyproterone,cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol,epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide,gemcitabine, gleevac, hydroxyurea, idarubicin, ifosfamide, imatinib,leuprolide, levamisole, lomustine, mechlorethamine, melphalan,6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane,mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate,pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab,streptozocin, teniposide, testosterone, thalidomide, thioguanine,thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalaninemustard, uracil mustard, estramustine, altretamine, floxuridine,5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin,calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine,topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291,squalamine, endostatin, SU541.6, SU6668, EMD1.21974, interleukin-12,IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone,finasteride, cimitidine, trastuzumab, denileukin diftitox, gefitinib,bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel,vinorelbine, bevacizumab (monoclonal antibody) and erbitux,cremophor-free paclitaxel, epithilone B, BMS-247550, BMS-310705,droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene,fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339,ZK186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycin,40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa and darbepoetin alfa, among others, and mixtures thereof.

In methods involving infections, disease states and/or conditions causedby rheumatoid arthritis, malaria, antiphospholipid antibody syndrome,lupus, chronic urticaria and Sjogren's disease, the compounds accordingto the present invention may be coadministered with additional agentswhich are traditionally used in therapy for these disease states and/orconditions.

Examples

The following examples illustrate and describe the present invention butare not intended to limit the invention in any way.

Chemical Methods

Strategy for Synthesis of Bivalent Aminoquinoline Autophagy Inhibitorswith Varying Linker Lengths

The compounds are prepared as previously described (PNAS) byBuchwald-Hartwig coupling of the appropriate spacer molecule diaminewith the requisite 4-substitutedquinoline, which are either commerciallyavailable or could be readily prepared by standard methods. To establishthe role of linker length, and of the asymmetric nature of the linkerlength of spermidine (3,4) relative to Lys01 (2,2), we have prepared aseries of new compounds that systematically vary the number ofmethylenes, i.e., CH2 groups, between the nitrogen atoms of the linker,where x and y=1-4 in the formula —HN(CH2)xNR(CH2)yNH—. These compoundsare listed in FIG. 10, Table 1, FIG. 11, Table 2 and FIG. 12 as Lys34-41 and Lys 72-75.

In addition, numerous compounds according to the present invention maybe readily prepared pursuant to the synthetic methods provided interalia on pages 21-28 of international application PCT/US2012/035251 (WO2012/149186), relevant portions of which are incorporated by referenceherein. Additional compounds may be prepared by analogy from thedisclosed methods as well as analogously from synthetic procedures whichare well known in the art.

Chemistry Experimental Protocol (Exemplary Syntheses)4-Bromo-7-chloroquinoline: J. Org. Chem. 2007 (72) 2232

At 0° C. under an atmosphere of argon, phosphorus tribromide (5.8 mL,0.079 moles, 1.10 equiv.) was added slowly to a solution of7-chloro-4-hydroxyquinoline (13.02 g, 0.073 moles, 1.00 equiv.) inanhydrous DMF (150 mL, 0.5 M soln.). The reaction was allowed to warm tort and followed by TLC. Complete consumption of starting material wasobserved after 90 minutes stirring. The reaction mixture was poured ontoice and the pH was rendered alkaline using solid sodium bicarbonate.This resulted in a white precipitate. The mixture was then filtered andthe resulting solid was dried under vacuum affording an off-white solid(17.30 g, 99%). The material was recrystallised from ethyl acetate togive white needles (12.20 g, 70%). R_(f)=0.70 (hex:EtoAc; 1:1);Mp=99-101° C., EtoAc (lit., Eur. J. Org. Chem. 2002 4181. 103-104° C.,hex).Lyso-01: McAfee et. al. PNAS

Under argon, a mixture of degassed dioxane and water (10:1; 22 mL, 0.4 Msoln.) was added to 4-bromo-7-chloroquinoline (4.78 g, 19.840 mmol, 2.25equiv.), BINAP (0.81 g, 1.302 mmol, 0.15 equiv.), Pd(OAc)₂ (0.16 g,0.716 mmol, 0.08 equiv.), potassium phosphate tribasic (6.911 g, 32.560mmol, 3.69 equiv.) and N-methylethane-1,2-diamine (0.94 mL, 8.814 mmol,1.00 equiv.). The reaction was heated to 120° C. in a sealed tube andheating was maintained for 16 hours. The reaction was then cooled to rtand filtered on a pad of celite washing through with chloroform (3×50mL). The reaction was rendered acidic via the addition of aqueous HCl (1M, 18 mL, 17.628 mmol, 2 equiv.) resulting in the precipitation of theHCl salt. This precipitate was isolated via filtration and the remainingorganic layer was extracted with deionised water (2×50 mL). The isolatedHCl salt was added to the aqueous washings which was then rendered basicvia the addition of ammonium hydroxide (until pH ≧9). This now alkalinemixture was extracted with CHCl₃ (3×40 mL). Combined chloroformfractions were washed with brine, dried over Na₂SO₄ and filtered.Concentration under reduced pressure resulted in the isolation of purecompound as an orange solid (2.74 g, 71%). The material wasrecrystallised from EtOAc, resulting in the isolation of needles (43%recrystallised yield). R_(f)=0.70.Lyso-05: McAfee et al. PNAS

A solution of the Lyso-01 (2.68 g, 6.098 mmol, 1.00 equiv.) in MeOH (20mL) was treated with 1M aqueous HCl (20 mL). The resulting reaction wasstirred vigorously at room temperature for 4 hours. The product wasinsoluble in the reaction media and was collected via filtration. Theresidue was collected and taken up in excess water. Sonication andheating was necessary to solubilise the product. The aqueous solutionwas lyophilised to give a white crystalline solid (1.77 g, 53%).Starting material (0.726 g, 27%) was recovered from the reaction.Mp=270° C. (decomposition) (Lit., PNAS 270° C.); HPLC analysis ofLyso-05 showed >99% purity.

Lyso-10:

Lyso-1 (0.26 g, 0.60 mmol, 1.00 equiv.) was placed under a blanket ofargon. Pyridine (0.5 mL, 5.95 mmol, 10.00 equiv.) followed by aceticanhydride (1.1 mL, 11.90 mmol, 20.00 equiv.) were added and theresulting reaction was heated to 150° C. for 2 hours. Following this thereaction mixture was poured onto a solvent mixture of CHCl₃ (40 mL) andwater (40 mL) and agitated vigorously. The resulting layers were allowedto settle and separated. The aqueous layer was further extracted withCHCl₃ (3×40 mL). Combined CHCl₃ extracts were washed with brine (120 mL)and dried over Na₂SO₄. Filtration followed by solvent evaporationafforded a brown oil (0.12 g, 38% yield) which was found to be verypure.

Lyso-20:

To a microwave vial was added spermine (0.57 g, 2.83 mmol, 1.00 equiv.),4-Bromo-7-chloroquinoline (1.32 g, 6.38 mmol, 2.25 equiv.), BINAP (0.11g, 0.17 mmol, 0.06 equiv.), Pd(OAc)₂ (0.02 g, 0.09 mmol, 0.03 equiv.)and K₃PO₄ (1.80 g, 8.50 mmol, 3.00 equiv.). The vial was sealed andplaced under argon. A solvent mixture of dioxane and water (10:1, 9 mL,0.3 M) was added and the reaction was heated to 120° C. for 16 hours.The reaction was followed by TLC and NMR and once complete it was cooledto room temperature. Following this the reaction was filtered on celite,washing through with MeOH (100 mL). The resulting solution wasconcentrated to give a solid. This solid was washed with CHCl₃ (3×30 mL)to remove impurities, affording the product as a yellowish solid (1.09g, 73% yield) which was found to be very pure.

Lyso-21:

To a sealable tube was added spermidine (0.98 g, 6.76 mmol, 1.00equiv.), 4-Bromo-7-chloroquinoline (3.69 g, 15.21 mmol, 2.25 equiv.),BINAP (0.17 g, 0.27 mmol, 0.04 equiv.), Pd(OAc)₂ (0.03 g, 0.14 mmol,0.02 equiv.) and K₃PO₄ (4.30 g, 20.28 mmol, 3.00 equiv.). The vial wassealed and placed under argon. A solvent mixture of dioxane and water(10:1, 22.5 mL, 0.3 M) was added and the reaction was heated to 120° C.for 16 hours. The reaction was followed by TLC and NMR and once completeit was cooled to room temperature. Following this the reaction wasfiltered on celite, washing through with MeOH (100 mL). The resultingsolution was concentrated to give a solid. This solid was solubilised inCHCl₃ (60 mL) and 1 M_(aq) HCl (60 mL) was added. The mixture wasstirred vigorously at room temperature for 30 minutes, affording thecorresponding water soluble HCl-salt of the product. The mixture wasallowed to settle and the resulting layers were separated. The aqueouslayer, containing the product, was further washed with CHCl₃ (2×60 mL).These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-propanol (4:1; 3×40 mL).These combined extracts were washed with brine (120 mL) and dried overNa₂SO₄. Filtration followed by solvent evaporation afforded the productas a white solid (2.05 g, 65% yield). R_(f)=0.10 (EtOAc:MeOH:TEA;80:15:5).

Lyso-25:

Lyso-20 (0.13 g, 0.25 mmol, 1.00 equiv.) was dissolved in DCM (2.5 mL,0.1 M). Formaldehyde 37 wt % (0.08 g, 0.98 mmol, 4.00 equiv.), thensodium triacetoxyborohydride (0.21 g, 0.98 mmol, 4.00 equiv.) was addedand the reaction was stirred at room temperature for 4 hours. At thispoint, TLC analysis showed complete consumption of Lyso-20. A 2 M_(aq)NaOH solution (15 mL) was added to break up borane salts in the reactionmixture. The resulting mixture was stirred at rt for 1 hour before CHCl₃(20 mL) was added. The resulting biphasic mixture was allowed to settleand separated. The aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_(aq) HCl solution (20 mL). Themixture was stirred vigorously at room temperature for 20 minutes,resulting in the formation of a water soluble salt of the desiredproduct. The mixture was allowed to settle and both layers wereseparated. The aqueous layer was further washed with CHCl₃ (2×40 mL) andcombined chloroform washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-propanol (4:1; 3×40 mL).These combined extracts were washed with brine (120 mL) and dried overNa₂SO₄. Filtration followed by solvent evaporation afforded the productas a white solid (16.0 mg, 12% yield). R_(f)=0.10 (EtOAc:MeOH:TEA;80:15:5).

Lyso-26:

Lyso-21 (0.11 g, 0.23 mmol, 1.00 equiv.) was dissolved in DCM (2.5 mL,0.1 M). Formaldehyde 37 wt % (0.04 g, 0.46 mmol, 2.00 equiv.), thensodium triacetoxyborohydride (0.19 g, 0.90 mmol, 4.00 equiv.) was addedand the reaction was stirred at room temperature for 4 hours. At thispoint, TLC analysis showed complete consumption of Lyso-21. A 2 M_(aq)NaOH solution (15 mL) was added to break up borane salts in the reactionmixture. The resulting mixture was stirred at rt for 1 hour before CHCl₃(20 mL) was added. The resulting biphasic mixture was allowed to settleand separated. The aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_(aq) HCl solution (15 mL). Themixture was stirred vigorously at room temperature for 20 minutes,resulting in the formation of a water soluble salt of the desiredproduct. The mixture was allowed to settle and both layers wereseparated. The aqueous layer was further washed with CHCl₃ (2×15 mL) andcombined chloroform washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-propanol (4:1; 3×15 mL).These combined extracts were washed with brine (60 mL) and dried overNa₂SO₄. Filtration followed by solvent evaporation afforded the productas a white solid (86.0 mg, 79% yield). R_(f)=0.30 (EtOAc:MeOH:TEA;80:15:5).

Ethyl (E)-3-((4-methoxyphenyl)amino)but-2-enoate

Prepared pursuant to Tetrahedron, 2012, 5522.

6-Methoxy-2-methylquinolin-4-ol

Prepared pursuant to Euro. J. Med. Chem. 2010, 3803.

4-Chloro-6-methoxy-2-methylquinoline: Euro. J. Med. Chem. 2010, 3803

Prepared pursuant to Euro. J. Med. Chem. 2010, 3803.

Lyso-27:

To a 20 mL microwave vial was added 1,9-diamino nonane (0.34 g, 2.18mmol, 1.00 equiv.), 4-Chloro-6-methoxy-2-methylquinoline (1.02 g, 4.90mmol, 2.25 equiv.), BINAP (0.07 g, 0.12 mmol, 0.05 equiv.), Pd(OAc)₂(0.02 g, 0.07 mmol, 0.03 equiv.) and K₃PO₄ (1.39 g, 6.53 mmol, 3.00equiv.). The vial was sealed and placed under argon. Fully degassessolvent dioxane:water (10:1, 7 mL, 0.3 M) was added and the reaction washeated to 100° C. for 16 hours. The reaction was followed by TLC andNMR. The reaction was allowed to cool to room temperature and filteredon celite, washing through with MeOH (100 mL). The filtrate wasconcentrated to give 0.87 g (crude yield 80%) of an impure brown solid.This material was purified via flash column chromatography (eluent:EtOAc:MeOH:TEA; 90:9:1) to give the product as an off-white solid (0.43g, 40% yield). R_(f)=0.25 (EtOAc:MeOH:TEA; 90:9:1).

Lyso-28:

Lyso-20 (0.32 g, 0.62 mmol, 1.00 equiv.) was suspended in DCM (6 mL, 0.1M). Freshly distilled benzaldehyde (0.4 mL, 3.69 mmol, 6.00 equiv) thensodium triacetoxyborohydride (1.30 g, 6.15 mmol, 10.00 equiv.) wereadded and the reaction was stirred at room temperature for 12 hours,until TLC analysis showed complete consumption of Lyso-20. A 2 M_(aq)NaOH solution (30 mL) was then added to break up borane salts in thereaction mixture. The resulting mixture was stirred at rt for 1 hourbefore DCM (30 mL) was added. The resulting biphasic mixture was allowedto settle and separated. The aqueous layer was discarded at this point.

To the DCM solution was added 1 M_(aq) HCl solution (30 mL) The mixturewas stirred vigorously at room temperature for 20 minutes, resulting inthe formation of a water soluble salt of the desired product. Themixture was allowed to settle and both layers were separated. Theaqueous layer was further washed with CHCl₃ (2×30 mL) and combined DCMwashings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of DCM (3×30 mL). These combinedextracts were washed with brine (100 mL) and dried over Na₂SO₄.Filtration followed by solvent evaporation afforded a yellow oil (0.35g, 80% crude yield) which contained impurities. This oil was purifiedvia flash column chromatography [gradient: 100% EtOAc (150 mL); thenEtOAc:MeOH:TEA; 90:9:1] to give the product as a yellow solid (0.21 g,49% yield). R_(f)=0.30 (EtOAc:MeOH:TEA; 90:9:1).

N-(2-Cyanoethyl)-N-(3-cyanopropyl)-benzylamine

Prepared pursuant to the procedure of J. Org. Chem. 1980, 1589.

N-(3-aminobutyl)-N-(3-aminopropyl)(phenylmethyl)amine: J. Org. Chem.1980, 1589

Prepared pursuant to the procedure of J. Org. Chem. 1980, 1589.

Lyso-29:

To a microwave tube was addedN-(3-aminobutyl)-N-(3-aminopropyl)(phenylmethyl)amine (96.00 mg, 0.41mmol, 1.00 equiv.), 4-Bromo-7-chloroquinoline (222.00 mg, 0.92 mmol,2.25 equiv.), BINAP (10.00 mg, 0.02 mmol, 0.04 equiv.), Pd(OAc)₂ (2.00mg, 0.01 mmol, 0.02 equiv.) and K₃PO₄ (260.00 mg, 1.22 mmol, 3.00equiv.). The vial was sealed and placed under argon. A solvent mixtureof dioxane and water (10:1, 1.5 mL, 0.3 M) was added and the reactionwas heated to 100° C. for 13 hours. The reaction was followed by TLC andNMR and once complete it was cooled to room temperature. Following thisthe reaction was filtered on celite, washing through with MeOH (20 mL).The resulting solution was concentrated to give a yellow oil. This oilwas solubilised in CHCl₃ (20 mL) and 1 M_(aq) HCl (20 mL) was added. Themixture was stirred vigorously at room temperature for 30 minutes,affording the corresponding water soluble HCl-salt of the product. Themixture was allowed to settle and the resulting layers were separated.The aqueous layer, containing the product, was further washed with CHCl₃(2×20 mL). These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into CHCl₃ (3×20 mL). These combined extracts were washed withbrine (60 mL) and dried over Na₂SO₄. Filtration followed by solventevaporation afforded the product as a yellow solid (112.00 mg, 49%yield). R_(f)=0.60 (EtOAc:MeOH:TEA; 80:15:5).

Lyso-30:

Under argon, Lyso-20 (0.25 g, 0.47 mmol, 1.00 equiv.) was dissolved inanhydrous pyridine (4.7 mL, 0.1 M) and freshly distilled benzoylchloride (0.3 mL, 2.37 mmol, 5.00 equiv.) was added. The reaction wasstirred at room temperature for 2 hours, until TLC analysis showedcomplete consumption of Lyso-20. Water (20 mL) and CHCl₃ (20 mL) werethen added to the reaction mixture and stirring was maintained for afurther 30 minutes. The layers of this biphasic mixture were thenseparated. Following this the aqueous layer was further extracted withCHCl₃ (2×10 mL). At this point the aqueous layer was discarded and theCHCl₃ extracts were combined.

To the CHCl₃ solution (containing the product) was added 1 M_(aq) HCl(40 mL). The resulting mixture was stirred vigorously at roomtemperature for 30 minutes, resulting in the formation of thecorresponding water soluble HCl-salt of the product. The mixture wasallowed to settle and the resulting layers were separated. The aqueouslayer, containing the product, was further washed with CHCl₃ (2×20 mL).These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into CHCl₃ (3×15 mL). These combined extracts were washed withbrine (60 mL) and dried over Na₂SO₄. Filtration followed by solventevaporation afforded the product as a white foam (0.13 g, 37% yield).R_(f)=0.40 (EtOAc:MeOH:TEA; 80:15:5).

Lyso-31:

Under argon, Lyso-21 (0.21 g, 0.45 mmol, 1.00 equiv.) was dissolved inanhydrous pyridine (4.5 mL, 0.1 M) and freshly distilled benzoylchloride (0.3 mL, 2.27 mmol, 5.00 equiv.) was added. The reaction wasstirred at room temperature for 2 hours, until TLC analysis showedcomplete consumption of Lyso-21. Water (10 mL) and CHCl₃ (10 mL) werethen added to the reaction mixture and stirring was maintained for afurther 30 minutes. The layers of this biphasic mixture were thenseparated. Following this the aqueous layer was further extracted withCHCl₃ (2×10 mL). At this point the aqueous layer was discarded and theCHCl₃ extracts were combined.

To the CHCl₃ solution (containing the product) was added 1 M_(aq) HCl(30 mL). The resulting mixture was stirred vigorously at roomtemperature for 30 minutes, resulting in the formation of thecorresponding water soluble HCl-salt of the product. The mixture wasallowed to settle and the resulting layers were separated. The aqueouslayer, containing the product, was further washed with CHCl₃ (2×10 mL).These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into CHCl₃ (3×15 mL). These combined extracts were washed withbrine (60 mL) and dried over Na₂SO₄. Filtration followed by solventevaporation afforded the 0.20 g (77% crude yield) of a red oil, whichcontained impurities. The material obtained was purified via flashcolumn chromatography (eluent: EtOAc:MeOH:TEA; 84:15:1) to give theproduct as an orange solid (0.14 g, 55% yield). R_(f)=0.45(EtOAc:MeOH:TEA; 80:15:5).

N-(tert-butylcarbonyl)-3-hydroxypropylamine

Prepared pursuant to the procedure of J. Med. Chem. 2009, 7029.

N-(tert-butylcarbonyl)-3-amino-propionaldehyde

Prepared pursuant to the procedure of J. Med. Chem. 2009, 7029.

Compound XX1

Lyso-21 (0.30 g, 0.64 mmol, 1.00 equiv.) was dissolved in DCM (13 mL,0.05 M). N-(tert-butylcarbonyl)-3-amino-propionaldehyde (0.33 g, 1.92mmol, 3.00 equiv.), then sodium triacetoxyborohydride (0.73 g, 3.84mmol, 6.00 equiv.) was added and the reaction was stirred at roomtemperature for 12 hours. At this point, TLC analysis showed completeconsumption of Lyso-21. A 2 M_(aq) NaOH solution (30 mL) was added tobreak up borane salts in the reaction mixture. The resulting mixture wasstirred at rt for 1 hour before DCM (20 mL) was added. The resultingbiphasic mixture was allowed to settle and separated. The aqueous layerwas discarded at this point.

To the DCM solution was added 1 M_(aq) HCl solution (30 mL). The mixturewas stirred vigorously at room temperature for 20 minutes, resulting inthe formation of a water soluble salt of the desired product. Themixture was allowed to settle and both layers were separated.

The aqueous layer was further washed with DCM (2×30 mL) and combined DCMwashings were then discarded.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into DCM (3×30 mL). These combined extracts were washed withbrine (100 mL) and dried over Na₂SO₄. Filtration followed by solventevaporation afforded a white foam which contained some impurities. Thisfoam was purified via flash column chromatography (eluent: gradient,100% EtOAc→EtOAc:MeOH:TEA; 80:15:5) to give the product as a white solid(0.23 g, 57% yield). R_(f)=0.50 (EtOAc:MeOH:TEA; 80:15:5).

Compound XX2

A solution of the compound from above (Compound XX1) (0.21 g, 0.34 mmol,1.00 equiv.) in DCM (3.5 mL, 0.1 M) was treated with TFA (2 mL, 26.118mmol, 77.00 equiv.). The resulting reaction was stirred at roomtemperature and followed by TLC. After 4 hrs, the reaction was found tohave reached completion and was quenched via the addition of saturatedNaHCO₃ solution. Once the reaction mixture was neutralised, it wasfurther basified to pH 11 using 2 M_(aq) NaOH solution. The now alkalinemixture was extracted with a solvent mixture of CHCl₃ and i-PrOH (4:1;3×40 mL). Combined organic extracts were washed with brine (100 mL) anddried over Na₂SO₄. Filtration followed by solvent evaporation affordedthe product as a yellow solid (0.13 g, 75% yield). R_(f)=0.00(EtOAc:MeOH:TEA; 80:15:5).

Lyso-32:

To a microwave tube was added compound XX2 (133.00 mg, 0.25 mmol, 1.00equiv.), 4-Bromo-7-chloroquinoline (86.00 mg, 0.35 mmol, 1.40 equiv.),BINAP (8.00 mg, 0.01 mmol, 0.05 equiv.), Pd(OAc)₂ (2.00 g, 0.008 mmol,0.03 equiv.) and K₃PO₄ (107.00 g, 0.51 mmol, 2.00 equiv.). The vial wassealed and placed under argon. A solvent mixture of dioxane and water(10:1, 1.0 mL, 0.3 M) was added and the reaction was heated to 120° C.for 13 hours. The reaction was followed by TLC and NMR and once completeit was cooled to room temperature. Following this the reaction wasfiltered on celite, washing through with MeOH (50 mL) The resultingsolution was concentrated to give 223.00 mg of a brown solid. This solidwas solubilised in CHCl₃ (30 mL) and 1 M_(aq) HCl (30 mL) was added. Themixture was stirred vigorously at room temperature for 30 minutes,affording the corresponding water soluble HCl-salt of the product. Themixture was allowed to settle and the resulting layers were separated.The aqueous layer, containing the product, was further washed with CHCl₃(2×30 mL). These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-PrOH (4:1; 3×50 mL).These combined extracts were washed with brine (150 mL) and dried overNa₂SO₄. Filtration followed by solvent evaporation afforded 155.00 mg(89% crude yield) of a white foam which contained some startingmaterial. This foam was purified via flash column chromatography(eluent: gradient EtOAc:MEOH:TEA; 90:9:1→EtOAc:MeOH:TEA; 85:15:5) togive the product as an orange solid (77.00 mg, 44% yield). R_(f)=0.60(EtOAc:MeOH:TEA; 80:15:5).

Compound XX3

Lyso-20 (0.23 g, 0.43 mmol, 1.00 equiv.) was dissolved in DCM (9 mL,0.05 M). N-(tert-butylcarbonyl)-3-amino-propionaldehyde (0.50 g, 2.86mmol, 6.00 equiv.), then sodium triacetoxyborohydride (0.91 g, 4.31mmol, 10.00 equiv.) was added and the reaction was stirred at roomtemperature for 12 hours. At this point, TLC analysis showed completeconsumption of Lyso-20. A 2 M_(aq) NaOH solution (30 mL) was added tobreak up borane salts in the reaction mixture. The resulting mixture wasstirred at rt for 1 hour before DCM (20 mL) was added. The resultingbiphasic mixture was allowed to settle and separated. The aqueous layerwas discarded at this point.

To the DCM solution was added 1 M_(aq) HCl solution (30 mL). The mixturewas stirred vigorously at room temperature for 20 minutes, resulting inthe formation of a water soluble salt of the desired product. Themixture was allowed to settle and both layers were separated. Theaqueous layer was further washed with DCM (2×30 mL) and combined DCMwashings were then discarded.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into DCM (3×30 mL). These combined extracts were washed withbrine (100 mL) and dried over Na₂SO₄. Filtration followed by solventevaporation afforded the product as a white foam (0.29 g, 81% yield).R_(f)=0.50 (EtOAc:MeOH:TEA; 80:15:5).

Compound XX4

A solution of Compound XX3 (0.28 g, 0.33 mmol, 1.00 equiv.) in DCM (3.3mL, 0.1 M) was treated with TFA (2 mL, 26.118 mmol, 79.00 equiv.). Theresulting reaction was stirred at room temperature and followed by TLC.After 4 hrs, the reaction was found to have reached completion and wasquenched via the addition of saturated NaHCO₃ solution. Once thereaction mixture was neutralised, it was further basified to pH 11 using2 M_(aq) NaOH solution. The now alkaline mixture was extracted with asolvent mixture of CHCl₃ and i-PrOH (4:1; 3×40 mL). Combined organicextracts were washed with brine (100 mL) and dried over Na₂SO₄.Filtration followed by solvent evaporation afforded the product as ayellow solid (0.09 g, 42% yield). R_(f)=0.00 (EtOAc:MeOH:TEA; 80:15:5).

Lyso-33:

To a microwave tube was added compound XX3 (90.00 mg, 0.14 mmol, 1.00equiv.), 4-Bromo-7-chloroquinoline (48.00 mg, 0.20 mmol, 1.40 equiv.),BINAP (4.00 mg, 0.007 mmol, 0.05 equiv.), Pd(OAc)₂ (1.00 mg, 0.004 mmol,0.03 equiv.) and K₃PO₄ (60.00 mg, 0.28 mmol, 2.00 equiv.). The vial wassealed and placed under argon. A solvent mixture of dioxane and water(10:1, 0.5 mL, 0.3 M) was added and the reaction was heated to 120° C.for 13 hours. The reaction was followed by TLC and NMR and once completeit was cooled to room temperature. Following this, the reaction wasfiltered on celite, washing through with MeOH (50 mL). The resultingsolution was concentrated to give 139.00 mg of a brown solid. This solidwas solubilised in CHCl₃ (30 mL) and 1 M_(aq) HCl (30 mL) was added. Themixture was stirred vigorously at room temperature for 30 minutes,affording the corresponding water soluble HCl-salt of the product. Themixture was allowed to settle and the resulting layers were separated.The aqueous layer, containing the product, was further washed with CHCl₃(2×30 mL). These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-PrOH (4:1; 3×50 mL).These combined extracts were washed with brine (150 mL) and dried overNa₂SO₄. Filtration followed by solvent evaporation afforded 85.00 mg(63% crude yield) of a brown foam which contained some startingmaterial. This foam was purified via flash column chromatography(eluent: CHCl₃:MeOH:TEA; 85:15:5) to give the product as a white solid(49.00 mg, 36% yield). R_(f)=0.55 (CHCl₃:MeOH:TEA; 80:15:5).

N,N-Bis(3-cyanopropyl)-benzylamine

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

N,N-Bis(4-aminobutyl)-benzylamine

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

Bis-(4-aminobutyl)-amine

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

Lyso-38:

To a microwave tube was added bis-(4-aminobutyl)-amine (0.31 g, 1.96mmol, 1.00 equiv.), 4-bromo-7-chloroquinoline (1.06 g, 4.41 mmol, 2.25equiv.), BINAP (0.09 g, 0.15 mmol, 0.08 equiv.), Pd(OAc)₂ (0.02 g, 0.10mmol, 0.05 equiv.) and K₃PO₄ (1.90 g, 7.84 mmol, 4.00 equiv.). The vialwas sealed and placed under argon. A solvent mixture of dioxane andwater (10:1, 6.5 mL, 0.3 M) was added and the reaction was heated to120° C. for 12 hours. The reaction was followed by TLC and NMR and oncecomplete it was cooled to room temperature. Following this, the reactionwas filtered on celite, washing through with MeOH (50 mL). The resultingsolution was concentrated under reduced pressure. This solid wassolubilised in DCM (40 mL) and 1 M_(aq) HCl (40 mL) was added. Themixture was stirred vigorously at room temperature for 30 minutes,affording the corresponding water soluble HCl-salt of the product. Themixture was allowed to settle and the resulting layers were separated.The aqueous layer, containing the product, was further washed with CHCl₃(2×40 mL). These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-PrOH (4:1; 3×40 mL).These combined extracts were washed with brine (120 mL) and dried overNa₂SO₄. Filtration followed by solvent evaporation afforded the productas a white solid (0.44 g, 46% yield). R_(f)=0.10 (EtOAc:MeOH:TEA;80:15:5).

Lyso-39:

A solution of Lyso-38 (0.13 g, 0.27 mmol, 1.00 equiv.) in DCM (3 mL, 0.1M) was treated with formaldehyde 37 wt % (0.04 g, 0.55 mmol, 2.00equiv.) then sodium triacetoxyborohydride (0.23 g, 1.09 mmol, 4.00equiv.). The resulting reaction was stirred at room temperature for 6hours. Following this, 2 M_(aq) NaOH (15 mL) was added to the reactionmixture to break up any borane salts. This mixture was stirred at roomtemperature for 1 hour before the product was extracted into CHCl₃ (3×15mL). The basic aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_(aq) HCl (40 mL). The mixturewas stirred vigorously at room temperature for 30 minutes, affording thecorresponding water soluble HCl-salt of the product. The mixture wasallowed to settle and the resulting layers were separated. The aqueouslayer, containing the product, was further washed with CHCl₃ (2×15 mL).These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-PrOH (4:1; 3×15 mL).These combined extracts were washed with brine (50 mL) and dried overNa₂SO₄. Filtration followed by solvent evaporation afforded the productas a white solid (0.08 g, 60% yield). R_(f)=0.30 (EtOAc:MeOH:TEA;80:15:5).

N,N-Bis(4-cyanobutyl)-benzylamine

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

N,N-Bis(5-aminopentyl)-benzylamine

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

Bis-(4-aminopentyl)-amine: Bioorg. Med. Chem. Lett. 2003. 3267

Prepared by the method described in Bioorg. Med. Chem. Lett. 2003. 3267.

Lyso-40:

To a microwave tube was added Bis-(4-aminopentyl)-amine (0.40 g, 1.43mmol, 1.00 equiv.), 4-bromo-7-chloroquinoline (0.78 g, 3.23 mmol, 2.25equiv.), BINAP (0.07 g, 0.11 mmol, 0.08 equiv.), Pd(OAc)₂ (0.02 g, 0.07mmol, 0.05 equiv.) and K₃PO₄ (1.22 g, 5.74 mmol, 4.00 equiv.). The vialwas sealed and placed under argon. A solvent mixture of dioxane andwater (10:1, 5 mL, 0.3 M) was added and the reaction was heated to 120°C. for 12 hours. The reaction was followed by TLC and NMR and oncecomplete it was cooled to room temperature. Following this, the reactionwas filtered on celite, washing through with MeOH (50 mL). The resultingsolution was concentrated under reduced pressure. This solid wassolubilised in CHCl₃ (20 mL) and 1 M_(aq) HCl (20 mL) was added. Themixture was stirred vigorously at room temperature for 30 minutes,affording the corresponding water soluble HCl-salt of the product. Themixture was allowed to settle and the resulting layers were separated.The aqueous layer, containing the product, was further washed with CHCl₃(2×20 mL). These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-PrOH (4:1; 3×30 mL).These combined organic extracts were washed with brine (100 mL) anddried over Na₂SO₄. Filtration followed by solvent evaporation affordedthe product as a yellow solid (0.51 g, 69% yield). R_(f)=0.05(EtOAc:MeOH:TEA; 84:15:1).

Lyso-41:

A solution of Lyso-40 (0.45 g, 0.88 mmol, 1.00 equiv.) in DCM (9 mL, 0.1M) was treated with formaldehyde 37 wt % (0.14 g, 1.76 mmol, 2.00equiv.) then sodium triacetoxyborohydride (0.74 g, 3.51 mmol, 4.00equiv.). The resulting reaction was stirred at room temperature for 6hours. Following this, 2 M_(aq) NaOH (30 mL) was added to the reactionmixture to break up any borane salts. This mixture was stirred at roomtemperature for 1 hour before the product was extracted into CHCl₃ (3×30mL). The basic aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_(aq) HCl (40 mL). The mixturewas stirred vigorously at room temperature for 30 minutes, affording thecorresponding water soluble HCl-salt of the product. The mixture wasallowed to settle and the resulting layers were separated. The aqueouslayer, containing the product, was further washed with CHCl₃ (2×30 mL).These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-PrOH (4:1; 3×30 mL).These combined extracts were washed with brine (100 mL) and dried overNa₂SO₄. Filtration followed by solvent evaporation afforded the productas a white solid (0.39 g, 85% yield). R_(f)=0.10 (EtOAc:MeOH:TEA;84:15:1).

Lys 42:

To a vial was added 4-Chloro-6-methoxy-2-methylquinoline (508.00 mg,2.45 mmol, 2.40 equiv), BINAP (95.00 mg, 0.15 mmol, 0.15 equiv.),Pd(OAc)₂ (17.00 mg, 0.08 mmol, 0.075 equiv.), finely ground K₃PO₄(867.00 mg, 4.09 mmol, 4.00 equiv.) andN-(2-aminoethyl)-N-methyl-1,2-ethanediamine (130.0 μL, 1.02 mmol, 1.00equiv.). The reagents were placed under a blanket of Argon; then amixture of degassed dioxane and water (4 mL, 10:1) was added. Thereaction vial was sealed and heated to 120° C. for 12 hours. Thereaction was then cooled to room temperature and filtered on a pad ofcelite; washing with chloroform (3×10 mL). Combined organic layers wereacidified to pH 1 using 1M aqueous HCl solution (4 mL, 4.09 mmol, 4.00equiv.) and diluted with water (20 mL). The resulting biphasic mixturewas separated and the water layer was washed with chloroform (2×15 mL).The pH of the aqueous layer was adjusted to 11 using ammonium hydroxide.The now alkaline mixture was washed with chloroform (3×20 mL). These 3chloroform extracts were combined, washed with brine, dried over Na₂SO₄and concentrated to give the product as an off-white solid (415.00 mg,88% yield, ≧95% purity). Recrystallisation from EtOAc afforded off-whitecrystals (0.225 g, 48% recrystallised yield). R_(f)=0.45(CHCl₃:MeOH:TEA; 85:10:5); Mp=205-206° C. with decomp. (EtOAc).

Lys 43:

A solution of Lyso-42 (1 equiv.) in DCM was treated with a 1 M solutionof HCl in diethyl ether (10 equiv.). HPLC analysis of Lyso-43showed >99% purity.

Lys 44:

To a vial was added 4-Chloro-6-methoxy-2-methylquinoline (559.00 mg,2.70 mmol, 2.40 equiv), BINAP (105.00 mg, 0.17 mmol, 0.15 equiv.),Pd(OAc)₂ (19.00 mg, 0.08 mmol, 0.075 equiv.), finely ground K₃PO₄(1061.00 mg, 5.00 mmol, 4.00 equiv.) and spermidine (180.0 μL, 1.13mmol, 1.00 equiv.). The reagents were placed under a blanket of Argon;then a mixture of degassed dioxane and water (4 mL, 10:1) was added. Thereaction vial was sealed and heated to 120° C. for 12 hours. Thereaction was then cooled to room temperature and filtered on a pad ofcelite; washing with chloroform (3×10 mL). Combined organic layers wereacidified to pH 1 using 1M aqueous HCl solution (5 mL, 4.5 mmol, 4.00equiv.) and diluted with water (20 mL). The resulting biphasic mixturewas separated and the water layer was washed with chloroform (2×15 mL).The pH of the aqueous layer was adjusted to 11 using ammonium hydroxide.The now alkaline mixture was washed with chloroform (3×20 mL). These 3chloroform extracts were combined, washed with brine, dried over Na₂SO₄and concentrated to give the product as an off-white solid (549.00 mg,99% yield, ≧90% purity). Recrystallisation from EtOAc afforded off-whitecrystals (123 mg, 22% recrystallised yield). R_(f)=0.05 (CHCl₃:MeOH:TEA;85:10:5); Mp=198 199° C. with decomp.(EtOAc).

Lys 45:

A solution of Lyso-44 (1 equiv.) in DCM was treated with a 1 M solutionof HCl in diethyl ether (10 equiv.). HPLC of Lyso-45 analysisshowed >97% purity.

Lys 46:

To a stirred mixture of Lys 44 (272.00 mg, 0.56 mmol, 1.00 equiv.) informic acid (2.2 mL, 0.25 M) was added 37% aqueous formaldehyde (99.8mg, 1.23 mmol, 2.20 equiv.). The reaction was heated to 100° C. andheating was maintained for 2 hours. TLC analysis revealed that all thestarting material had been consumed. The reaction mixture was pouredonto ice and basified via the addition of ammonium hydroxide (until pH≧9). The now alkaline mixture was extracted with chloroform (3×15 mL).Combined organic extracts were washed with brine and dried over Na₂SO₄.Filtration followed by solvent evaporation afforded the product as anoff-white solid (261.00 mg, 93% yield, ≧85% purity). The material waspurified via flash column chromatography (gradient: 100% EtOAc→84:15:1;EtOAc:MeOH:TEA) to give a pure off-white solid (102.00 mg, 36% yield).R_(f)=0.15 (CHCl₃:MeOH:TEA; 85:10:5); Mp=140-141° C.

Lys 47:

A solution of Lyso-46 (1 equiv.) in DCM was treated with a 1 M solutionof HCl in diethyl ether (10 equiv.). HPLC analysis of Lyso-47showed >99% purity.

Lyso-72:

To a microwave tube was added Bis-(hexamethyl)-triamine (0.73 g, 3.40mmol, 1.00 equiv.), 4-bromo-7-chloroquinoline (1.86 g, 7.66 mmol, 2.25equiv.), BINAP (0.11 g, 0.17 mmol, 0.05 equiv.), Pd(OAc)₂ (0.02 g, 0.10mmol, 0.03 equiv.) and K₃PO₄ (2.17 g, 10.21 mmol, 3.00 equiv.). The vialwas sealed and placed under argon. A solvent mixture of dioxane andwater (10:1, 11 mL, 0.3 M) was added and the reaction was heated to 120°C. for 14 hours. The reaction was followed by TLC and NMR and oncecomplete it was cooled to room temperature. Following this, the reactionwas filtered on celite, washing through with MeOH (50 mL). The resultingsolution was concentrated under reduced pressure. This solid wassolubilised in CHCl₃ (50 mL) and 1 M_(aq) HCl (50 mL) was added. Themixture was stirred vigorously at room temperature for 30 minutes,affording the corresponding water soluble HCl-salt of the product. Themixture was allowed to settle and the resulting layers were separated.The aqueous layer, containing the product, was further washed with CHCl₃(2×50 mL). These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-PrOH (4:1; 3×40 mL).These combined organic extracts were washed with brine (120 mL) anddried over Na₂SO₄. Filtration followed by solvent evaporation affordedthe product as a yellow solid (1.66 g, 90% yield). R_(f)=0.10(EtOAc:MeOH:TEA; 80:15:5); Mp=100-102° C.

Lyso-73:

A solution of Lyso-72 (1 equiv.) in DCM was treated with a 1 M solutionof HCl in diethyl ether (10 equiv.). HPLC analysis of Lyso-73showed >99% purity.

Lyso-74:

A solution of Lyso-72 (0.93 g, 1.73 mmol, 1.00 equiv.) in THF (17 mL,0.1 M) was treated with formaldehyde 37 wt % (0.28 g, 3.45 mmol, 2.00equiv.) then sodium triacetoxyborohydride (1.46 g, 6.91 mmol, 4.00equiv.). The resulting reaction was stirred at room temperature for 4hours. Following this, 2 M_(aq) NaOH (30 mL) was added to the reactionmixture to break up any borane salts. This mixture was stirred at roomtemperature for 1 hour before the product was extracted into CHCl₃ (30mL). The basic aqueous layer was discarded at this point.

To the chloroform solution was added 1 M_(aq) HCl (30 mL). The mixturewas stirred vigorously at room temperature for 30 minutes, affording thecorresponding water soluble HCl-salt of the product. The mixture wasallowed to settle and the resulting layers were separated. The aqueouslayer, containing the product, was then further washed with CHCl₃ (2×30mL). These combined organic washings were discarded at this point.

Using NH₄.OH, the pH of the aqueous layer was adjusted to 11, resultingin the liberation of the free base of the product. The product wasextracted into a solvent mixture of CHCl₃ and i-PrOH (4:1; 3×30 mL).These combined extracts were washed with brine (120 mL) and dried overNa₂SO₄. Filtration followed by solvent evaporation afforded the productas a yellow solid (0.34 g, 36% yield). R_(f)=0.35 (EtOAc:MeOH:TEA;80:15:5); Mp=58-60° C.

Lyso-75:

A solution of Lyso-74 (1 equiv.) in DCM was treated with a 1 M solutionof HCl in diethyl ether (10 equiv.). HPLC analysis of Lyso-75showed >99% purity.

Lyso-82:

To a 20 mL microwave vial was added 1,9-diamino nonane (0.27 g, 1.72mmol, 1.00 equiv.), 4-bromo-7-chloroquinoline (1.00 g, 4.12 mmol, 2.40equiv.), BINAP (0.05 g, 0.09 mmol, 0.05 equiv.), Pd(OAc)₂ (0.01 g, 0.05mmol, 0.03 equiv.) and K₃PO₄ (1.09 g, 5.15 mmol, 3.00 equiv.). The vialwas sealed and placed under argon. Fully degasses solvent dioxane:water(10:1, 6 mL, 0.3 M) was added and the reaction was heated to 120° C. for12 hours. The reaction was followed by TLC and NMR. The reaction wasallowed to cool to room temperature and filtered on celite, washingthrough with MeOH (100 mL). The filtrate was concentrated to give 1.09 g(crude yield 131%) of an impure solid. This material was purified viaflash column chromatography (eluent: Gradient; 100% EtOAcEtOAc:MeOH:TEA; 90:9:1) to give the product as a white solid (0.40 g,37% yield). R_(f)=0.40 (EtOAc:MeOH:TEA; 90:9:1); Mp=158-160° C.

Lyso-83:

A solution of Lyso-82 (1 equiv.) in DCM was treated with a 1 M solutionof HCl in diethyl ether (10 equiv.). HPLC analysis of Lyso-83showed >90% purity.

Results

The present analogs have been designed to test an important factor: therole of the linker between the two chloroquinoline moieties. Toward thatend, we have observed that replacement of the linker of Lys01 withlonger linkers, such as spermine (Lys 20 and 25) and spermidine (Lys21and 26) lead to significant increases in potency. While the sperminelinker introduces an additional nitrogen functionality (and anadditional positive charge under physiological conditions), thespermidine linker is more similar to the Lys01 linker. However, unlikethe Lys01 linker, the spermidine linker is not symmetrical. Thespermidine linker contains three and four methylenes, respectively,between the three nitrogen atoms of the triamine linker, while the Lys01connector contains two methylenes between each of the three nitrogenatoms.

Cytotoxic Activity of Longer and Assymetric Linker Compounds in CancerCells.

LN229 glioma cells, and A375P melanoma cells were plated in a 384 wellformat and compounds CQ, HCQ, and Lys01-Lys41 and 72-75 (structuresprovided in Appendix A, Chemical Structures) were delivered inconcentrations between 0.01-10 micromolar using robotic assisteddispenser. After 72 hours incubation at 37 degrees, Alamar blue wasapplied and viability was determined using absorbance. Absorbance wasnormalized to DMSO control, and a log IC50 was estimated using GraphpadPrism software (FIG. 11, Table 1). LN229 cells produced similar data(data not shown). Selected IC50s of the most promising compounds arepresented in FIG. 1A. The increased cytotoxicity of longer linker andassyemtric compounds was confirmed in a 2 week colony formation assayusing 5 different cell lines (FIG. 1B). Analysis of the cytotoxicityfindings alone elucidated some key features of potential structureactivity relationships. First, substitution of the central nitrogensubstitution beyond methyl reduces activity. Second, there was no clearadvantage of assymetric versus symmetric linkers (FIG. 2A) with regardsto cytotoxicity alone. Third, increasing Linker length from Lys05 wasassociated with increased activity but beyond a certain threshold oflinker carbons, there is no increased potency for these compounds in thealamar blue viability assay (FIG. 2B).

Longer Linker Compounds Localize to the Lysosome.

To confirm that dimeric CQ's with longer linkers retain their presumedlysosomal subcellular localization, Lys21/26 was functionalized into afluorescent probe by attaching a Cy3 dye to the central nitrogen. Thiscompound referred to as Lys21-Cy3 (FIG. 3A) was used to treat A375melanoma (FIG. 3B). Costaining with lysotracker green and subsequentfluorescent microscopy allowed the visualization of lysosomallocalization for the Lys21 derivative. After 48 hours of treatmentlysosomal mass had decreased and the red fluorescence was morecytoplasmic indicative of lysosomal rupture and diffusion of intactcompound.

Longer Linker Compounds are Associated with More Profound AutophagyModulation, Reactive Oxygen Species (ROS) and Apoptosis

As an initial method of determining the effects of the longer linkercompounds on autophagic vesicle accumulation, a series of most cytotoxicdimeric CQ's were used to treat 3 different cell lines (1 uM, 24 hours).Cell lysates were collected and immunoblotting against LC3 and actin asperformed (FIG. 4). To determine if increased cytotoxicity associatedwith Lys01 derivatives correlated with more potent inhibition ofautophagy, LC3 immunoblotting was performed. LC3 is a ubiquitin-likeprotein which exists as a unconjugated form (LC3I) or conjugated to AVmembranes (LC3II)(23). Quantification of LC3II/actin bands demonstratedthat all of the longer linker compounds were superior to Lys05 atmodulating LC3II levels. Although there was some variability acrossmelanoma cell lines, in all cases the longest linker compound Lys75produced the most consistent and substantial increase in the LC3II/actinration. To confirm that LC3II modulation observed was indeed due to theinhibition of autophagic flux, A375P mCherry-GFP-LC3 cells were used ina high throughput phenotypic screen. In cells expressing this tandemfluorescent reporter construct autophagy induction results inaccumulation of red puncta, because the green GFP fluorescence ispartially quenched when autophagic vesicles fuse with the lysosomes. Incells where autophagic flux is inhibited distally due to lysosomalimpairment, the green signal persists, turning puncta into a yellowcolor. Lys01-Lys27, HCQ, CQ and doxorubicin which serves as a controlwere administered to cells in concentrations ranging from 0-10 uM usingpinwheel transfer, Operetta confocal imaging allowed image capture forhigh content analysis (FIG. 5, FIG. 6). Simple analysis of per cell(based on nuclear Hoescht staining) basis demonstrated that at 10 μMconcentrations, compounds which had previously been identified as havinglower IC50 in viability experiments also produced green/red fluorescentratios>3.5. Histograms of % cells with green:red ratio>3.5 demonstratesthat in some cases lower doses of the compounds also produced thisphenotype indicative of effective autophagy inhibition (FIG. 7).Previous studies have demonstrated that CQ derivatives produce ROS incancer cells. Each of the longer linker Lys01 derivative was superior toLys05 in inducing ROS (FIG. 8A). Lys26 (closely related to Lys21) andLys75 produced the highest levels of ROS. Flow cytometry was used toquantify the green/red ration indicative of inhibition of autophagicflux in A375P mCHerry-eGFP-LC3 cells treated with longer linker dimericCQs (FIG. 8B). Again, Lys21 and Lys75 were most effective at inhibitingautophagic flux. Finally, to determine if this inhibition of autophagicflux had a functional consequence of cell death, annexin V whichreflects apoptosis was measured in A375 cells treated with compounds.Similar to the results observed with autophgic flux, Lys21 and Lys75produced the most apoptosis at the 24 hour timepoint (FIG. 8C). To putthis degree of apoptosis into clinical context, BRAF mutant melanomacells were treated with longer linker dimeric CQs or standard of careBRAF, or BRAF (PLX4720) and MEK inhibitor (trametinib). The longerlinker Lys01 derivatives produced significantly more apoptosis thanclinically approved kinase inhibitors despite equimolar dosing (FIG.8D).

Dimeric CQ's with Longer Linkers Impair Growth and Invasion of Melanomain a 3 Dimensional Tissue Like Culture System.

The 3D spheroid model has been used extensively to mimic the tumormicroenvironment (Ma et al Clin Can Research 2011). Autophagy levels aremore refelective of in vivo autophagy dynamics in the 3D models comparedto traditional two dimensional cell culture. In this format cancer cellsare grown as spheroids and then implanted in a collagen matrix. Thespheroids thn begin to invade and grow within this tissuemicroenvironment. Treatment with various longer linker dimericchloroquines once again demonstrated that Lys75 was the most potentcompound at inhibiting growth and invasion of the 3D spheroids (FIG. 9A,B).

Longer Linker Compounds

To further investigate the role of linker length with regards to potencywe prepare a series of compounds with even longer linkers (FIG. 13Compounds Lys86-Lys97). We also revisit the effects of quinolone ringsubstitution in the setting of these longer linked compound (FIG. 13).

Summary and Conclusion

Increasing linker length between quinolone rings in dimeric chloroquinessignificantly improved anti-cancer cytoxicity, ROS production, andautophagy inhibition. Assyemtric dimeric chloroquines showed similaranticancer cytotoxicity in 72 hour viability assays to comparablesymmetric counterparts. However in the case of Lys21, superior autophagyinhibition and ability to induce apoptosis was observed compared tocomparable compounds that were symmetric.

The GI toxicity associated with Paneth cell dysfunction observed at LD30doses of Lys05, support the mechanism of action of the presentinvention, and also suggests that colon cancers, which often sharefeatures with Paneth cells, may be a tumor type, among others, that maybe particularly sensitive to Lys05 and its optimized derivatives.Additional cancers which represent particularly important targetsinclude melanoma, and non-small cell lung cancer, since melanoma celllines demonstrated the highest difference in sensitivity to Lys01compared to HCQ, and an EGFR mutated lung cancer cell line demonstratedsensitivity to both HCQ and Lys05. Further mechanistic studies areplanned that will potentially identify pharmacodynamics assays thatguide drug development. Pharmacokinetic studies are planned in mice toestablish initial in vivo profile.

Advantages over other similar technologies: The present inventionrepresents the most potent bisaminoquinoline derivatives reported thusfar in cell lines. Other autophagy compounds are not focused on thelysosome as a target and consequently do not evidence the level ofactivity of compounds according to the present invention.

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1. A compound of Formula IA:

wherein R¹ and R^(1′) are each independently H, F, Cl, Br, I, CN, NO₂,optionally substituted C₁-C₆ alkyl, optionally substituted O—C₁-C₆alkyl, optionally substituted C₂-C₇ acyl, optionally substituted—(NH)-acyl, or optionally substituted C₂-C₇ ester; R and R′ are eachindependently H, a C₁-C₆ optionally substituted alkyl group, a C₁-C₇optionally substituted acyl group, a C₂-C₇ optionally substitutedcarboxy ester group (which forms a urethane group with the nitrogen atomto which R or R′ is bonded); R″ is H, Cy¹ or (C═O)_(z)-G, where Cy¹ isan optionally substituted cycloalkyl, aryl or heteroaryl group G is anoptionally substituted C₀-C₁₂ alkyl, alkene or alkynyl group (whereinoptional substituents include a C₁-C₁₂ alkyl, alkene or alkynyl groupsubstituted by (N—R^(J))—(C₁-C₈ alkyl, alkene or alkynylgroup)_(z)-(Cy²)_(x), where R^(J) is H or a C₁-C₈ alkyl, alkene oralkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is anoptionally substituted aryl or heteroaryl group); R^(M) is independentlyat each occurrence H, F, Cl, Br, I, an optionally substituted C₁-C₁₂alkyl, alkene, alkynyl or alkoxy group; Each p in linker group L^(1A) isindependently 1-10; and the pharmaceutically acceptable salts,enantiomers, diastereomers, solvates and polymorphs thereof.
 2. Acompound of Formula IB:

wherein R¹ and R^(1′) are each independently H, F, Cl, Br, I, CN, NO₂,optionally substituted C₁-C₆ alkyl, optionally substituted O—C₁-C₆alkyl, optionally substituted C₂-C₇ acyl, an optionally substituted—(NH)-acyl, or optionally substituted C₂-C₇ ester; R and R′ are eachindependently H, a C₁-C₆ optionally substituted alkyl group, a C₁-C₇optionally substituted acyl group, a C₂-C₇ optionally substitutedcarboxy ester group (which forms a urethane group with the nitrogen atomto which R or R′ is bonded); R″ is H, Cy¹ or (C═O)_(z)-G, where Cy¹ isan optionally substituted cycloalkyl, aryl or heteroaryl group G is anoptionally substituted C₀-C₁₂ alkyl, alkene or alkynyl group (whereinoptional substituents include a C₁-C₁₂ alkyl, alkene or alkynyl groupsubstituted by (N—R^(J))—(C₁-C₈ alkyl, alkene or alkynylgroup)_(z)-(Cy²)_(x), where R^(J) is H or a C₁-C₅ alkyl, alkene oralkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is anoptionally substituted aryl or heteroaryl group); R^(M) is independentlyat each occurrence H, F, Cl, Br, I, an optionally substituted C₀-C₁₂alkyl, alkene, alkynyl or alkoxy group; p and p′ in linker L^(1B) areeach independently 1-10 (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), and thepharmaceutically acceptable salts, enantiomers, diastereomers, solvatesand polymorphs thereof.
 3. A compound of Formula IC:

wherein R¹ and R^(1′) are each independently H, F, Cl, Br, I, CN, NO₂,optionally substituted C₁-C₆ alkyl, optionally substituted O—C₁-C₆alkyl, optionally substituted C₂-C₇ acyl, an optionally substituted—(NH)-acyl, or optionally substituted C₂-C₇ ester; R and R′ are eachindependently H, a C₁-C₆ optionally substituted alkyl group, a C₁-C₇optionally substituted acyl group, a C₂-C₇ optionally substitutedcarboxy ester group (which forms a urethane group with the nitrogen atomto which R or R′ is bonded); R″ is H, Cy¹ or (C═O)_(z)-G, where Cy¹ isan optionally substituted cycloalkyl, aryl or heteroaryl group G is anoptionally substituted C₀-C₁₂ alkyl, alkene or alkynyl group (whereinoptional substituents include a C₁-C₁₂ alkyl, alkene or alkynyl groupsubstituted by (N—R^(J))—(C₁-C₈ alkyl, alkene or alkynylgroup)_(z)-(Cy²)_(x), where R^(J) is H or a C₁-C₈ alkyl, alkene oralkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is anoptionally substituted aryl or heteroaryl group); R^(M) is independentlyat each occurrence H, F, Cl, Br, I, an optionally substituted C₀-C₁₂alkyl, alkene, alkynyl or alkoxy group; Each p in linker group L^(1C) isindependently 1-10 (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); and thepharmaceutically acceptable salts, enantiomers, diastereomers, solvatessand polymorphs thereof.
 4. A compound of Formula ID:

wherein R′ and R″ are each independently H, F, Cl, Br, I, CN, NO₂,optionally substituted C₁-C₆ alkyl, optionally substituted O—C₁-C₆alkyl, optionally substituted C₂-C₇ acyl, optionally substituted—(NH)-acyl, or optionally substituted C₂-C₇ ester; R and R′ are eachindependently H, a C₁-C₆ optionally substituted alkyl group, a C₁-C₇optionally substituted acyl group, a C₂-C₇ optionally substitutedcarboxy ester group (which forms a urethane group with the nitrogen atomto which R or R′ is bonded); R″ is H, Cy¹ or (C═O)_(z)-G, where Cy¹ isan optionally substituted cycloalkyl, aryl or heteroaryl group G is anoptionally substituted C₀-C₁₂ alkyl, alkene or alkynyl group (whereinoptional substituents include a C₁-C₁₂ alkyl, alkene or alkynyl groupsubstituted by (N—R^(J))—(C₁-C₈ alkyl, alkene or alkynylgroup)_(z)-(Cy²)_(x), where R^(J) is H or a C₁-C₈ alkyl, alkene oralkynyl group, z is 0, 1, 2, 3, 4 or 5, x is 0 or 1 and Cy² is anoptionally substituted aryl or heteroaryl group); R^(M) is independentlyat each occurrence H, F, Cl, Br, I, an optionally substituted C₀-C₁₂alkyl, alkene, alkynyl or alkoxy group; Linker group L^(1D) is anoptionally substituted alkylene group containing from 1-20 methylenegroups; and the pharmaceutically acceptable salts, enantiomers,diastereomers, solvates and polymorphs thereof.
 5. A compound of formulaIE:

Wherein R¹ and R^(1′) are each independently H, halo (F, Cl, Br or I),CN, NO₂, optionally substituted C₁-C₆ alkyl, optionally substitutedO—C₁-C₆ alkyl, optionally substituted —(NH)-acyl, optionally substitutedC₂-C₇ acyl or optionally substituted C₂-C₇ ester (oxycarbonyl ester orcarboxyester, preferably carboxyester); R^(M) is independently at eachoccurrence H, F, Cl, Br, I, an optionally substituted C₁-C₁₂ (preferablyC₁-C₈) alkyl, alkene, alkynyl or alkoxy group; R and R′ are eachindependently H, a C₁-C₆ optionally substituted alkyl group, a C₁-C₇(preferably C₂-C₇) optionally substituted acyl group, a C₂-C₇ optionallysubstituted carboxy ester group (which forms a urethane group with thenitrogen atom to which R or R′ is bonded); Linker group L^(1E) is a

group or a

group wherein at least one of the CH₂ groups in L^(1E) is optionallysubstituted with an optionally substituted C₁-C₆ alkyl group including acyclic alkyl group, each of which alkyl groups is optionallysubstituted, or at least two methylene groups (preferably two adjacentmethylene groups) are optionally substituted with alkylene groups toform an optionally substituted C₃-C₈ cycloalkyl group, or linker groupL^(1E) is a (poly)ethylene glycol group having from 1 to 20 ethyleneglycol units, each (poly)ethylene glycol group being optionallysubstituted at one or both of its distal ends with an A group, or L is alinear group containing from 1 to 5 non-contiguous amine groups N—R″,each amine group being separated from an adjacent amine group by analkylene group containing from 1, 2, 3, 4, 5, 6, 7, or 8 (preferably 2,3, 4, 5 or 6) methylene groups or an ethylene glycol containing grouphaving from 1 to 6 (preferably 2 to 6, 3 to 6 or 4, 5 or 6) ethyleneglycol units; X is absent, (CH₂)_(j) O, S or N—R″; Y is absent, CH₂, O,CH₂O or N—R″ and Y′ is absent CH₂, O, OCH₂ or N—R″, with the provisothat when one or more of X, Y and Y′ is present, each of X and Y, X andY′ or Y and Y′, when present, forms a stable bond; R″ is H or anoptionally substituted C₁-C₆ alkyl group or an amine protecting group;each j is independently 1, 2, 3, 4, 5 or 6; each n is independently 0,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 with the proviso that when n is 0, X is(CH₂)_(j) where j is at least 1 and at least one CH₂ group is optionallysubstituted, preferably with a C₁-C₃ alkyl group which itself isoptionally substituted with one or two hydroxyl groups or one, two, orthree halo groups; A is absent, C═O or (CH₂)_(j) and A′ is C═O or(CH₂)_(j) wherein at least one CH₂ group in A or A′ is optionallysubstituted, preferably with a C₁-C₃ alkyl group which is itselfoptionally substituted; Z is O or N—R^(Z); R^(Z) is H or an optionallysubstituted C₁-C₃ alkyl group, or a pharmaceutically acceptable salt,enantiomer, diastereomer, solvent or polymorph thereof.
 6. The compoundaccording to claim 5 wherein at least two R^(M) are other than H.
 7. Thecompound according to claim 5 wherein R¹ and R^(1′) are eachindependently H or a halo group (preferably F or Cl).
 8. The compoundaccording to claim 5 wherein L is an alkylene group containing from 6 to20 methylene groups (C₆-C₂₀ alkylene group).
 9. The compound accordingto claim 5 wherein L is a (poly)ethylene glycol group having from 1 to20 ethylene glycol units optionally substituted at one or both distalends of said (poly)ethylene glycol group with an A group.
 10. A compoundaccording to claim 5 wherein L is a linear group containing from 1 to 5non-contiguous amine groups N—R″, each amine group being separated froman adjacent amine group by an alkylene group containing 1, 2, 3, 4, 5,6, 7, or 8 methylene groups or an ethylene glycol containing groupcontaining 1, 2, 3, 4, 5 or 6 ethylene glycol units.
 11. A compound offormulae IIA, IIB, IIC or IID, and the pharmaceutically acceptablesalts, enantiomers, diastereomers, solvates or polymorphs thereof:

where X is a halogen, preferably F or Cl.
 12. A compound according toclaim 5 wherein X is Cl (Lys-74) or a pharmaceutically acceptable saltthereof.
 13. A compound selected from the group consisting of thecompounds listed in FIG. 10, 11, 12 or 13 herein, and thepharmaceutically acceptable salts, enantiomers, diastereomers, solvatesand polymorphs thereof.
 14. A pharmaceutical composition comprising atherapeutically effective amount of at least one compound according toclaim 1 in combination with a pharmaceutically acceptable carrier,additive or excipient.
 15. The composition according to claim 14comprising a compound of formulae IIA, IIB, IIC or IID, and thepharmaceutically acceptable salts, enantiomers, diastereomers, solvatesor polymorphs thereof:

where X is a halogen, preferably F or Cl.
 16. The composition accordingto claim 15 wherein said compound is Lys-21, Lys-25, Lys-2, Lys-75, amixture thereof or a pharmaceutical acceptable salt(s) thereof.
 17. Thecomposition according to claim 14 further comprising an effective amountof at least one additional anticancer agent.
 18. The compositionaccording to claim 17 wherein said anticancer agent is a FLT-3inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinaseinhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhbitor, ac-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TKinhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinaseinhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek)inhibitor, a VEGF trap antibody or a mixture thereof.
 19. Thecomposition according to claim 17 wherein said anticancer agent isselected from the group consisting of everolimus, trabectedin, abraxane,TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263,pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab,amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin,ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan,tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111,131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan,IL13-PE38QQR; INO 1001, IPdR₁ KRX-0402, lucanthone, LY 317615,neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311,romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat,etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin,5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709,seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid,N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-,disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan,tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated, estrogen,bevacizumab, IMC-1C11, CHIR-258);3-[5-(methylsulfonylpiperadinemethyl)-indolylj-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6, Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂)_(X) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714;TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662,tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid,valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951,aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BacillusCalmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevac,hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole,lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna,methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide,oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, teniposide,testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine,13-cis-retinoic acid, phenylalanine mustard, uracil mustard,estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosinearabinoside, 6-mecaptopurine, deoxycoformycin, calcitriol, valrubicin,mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat,COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668,EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene,idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab,denileukin diftitox, gefitinib, bortezimib, paclitaxel, irinotecan,topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonalantibody) and erbitux, cremophor-free paclitaxel, epithilone B,BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene,ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene,TSE-424, HMR-3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352,rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573,RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684,LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, sspegfilgrastim, erythropoietin,epoetin alfa and darbepoetin alfa, ipilumumab, vemurafenib and mixturesthereof.
 20. A method of inhibiting autophagy in a biological system inwhich inhibition of autophagy is desired, said method comprisingexposing said biological system to an effective amount of at least onecompound according to claim 1 hereof.
 21. A method of inhibiting ortreating cancer in a patient in need comprising administering to saidpatient an effective amount of at least one composition according toclaim
 14. 22. The method according to claim 21, wherein said cancer ismetastatic, recurrent and/or drug resistant.
 23. (canceled) 24.(canceled)
 25. A method of reducing the likelihood that cancer willoccur in a patient or that a cancer will metastasize in a patientcomprising administering at least one composition according to claim 14,optionally in combination with at least one additional anticancer agent.26. The method according to claim 21, wherein said cancer is acarcinoma, cancer of the esophagus, head, kidney, liver, lung,nasopharyngeal, neck, ovary, pancreas, prostate, and stomach; aleukemia, a malignant lymphoma, a malignant melanoma; myeloproliferativediseases; a sarcoma, a tumor of the central nervous system, a germ-linetumor, lung cancer, ovarian cancer, testicular cancer, thyroid cancer,astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, livercancer, colon cancer, melanoma, or a mixed type of neoplasia.
 27. Themethod according to claim 26, wherein said leukemia is acute myelogenousleukemia, acute lymphocytic leukemia, acute promyelocytic leukemia(APL), acute T-cell lymphoblastic leukemia, adult T-cell leukemia,basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairycell leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblasticleukemia, lymphocytic leukemia, megakaryocytic leukemia,micromyeloblastic leukemia, monocytic leukemia, neutrophilic leukemiaand stem cell leukemia, said lymphoma is Burkitt's lymphoma,Non-Hodgkin's lymphoma or B-cell lymphoma, said sarcoma is Ewing'ssarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas,peripheral neuroepithelioma or synovial sarcoma, said tumor of thecentral nervous system is a glioma, astrocytoma, oligodendroglioma,ependymoma, gliobastoma, neuroblastoma, ganglioneuroma, ganglioglioma,medulloblastoma, pineal cell tumor, meningioma, meningeal sarcoma,neurofibroma, or Schwannoma, said germ-line tumor is bowel cancer,breast cancer, prostate cancer, cervical cancer or uterine cancer, saidlung cancer is small cell lung cancer, mixed small cell and non-smallcell cancer, pleural mesothelioma, metastatic pleural mesothelioma,small cell lung cancer or non-small cell lung cancer, said mixedneoplasia is carcinosarcoma and Hodgkin's disease and said tumors ofmixed origin is Wilms' tumor and teratocarcinomas, said cancer isovarian, breast, colon, head and neck, medulloblastoma or B-celllymphoma, said cancer is melanoma or non-small cell lung cancer. 28.(canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled)33. (canceled)
 34. (canceled)
 35. (canceled)
 36. A method of treating adisease state or condition in a patient in need wherein said diseasestate or condition responds favorably to inhibition of autophagycomprising administering to said patient an effective amount of acompound according to claim 1 to said patient.
 37. The method accordingto claim 36, wherein said disease state or condition is rheumatoidarthritis, malaria, antiphospholipid antibody syndrome, lupus, chronicurticaria or Sjogren's disease.
 38. The method according to claim 37,wherein said disease state is malaria. 39.-59. (canceled)